Method for inducing differentiation of pluripotent stem cells into germ cells

ABSTRACT

The invention provides a method for inducing human primordial germ cell-like (PGC-like) cells from human pluripotent stem cells, with high efficiency and high reproducibility, and a cell surface marker for identifying human PGC-like cells. In particular, the invention provides a method for producing a human PGC-like cell from a human pluripotent stem cell, includes a step of producing a mesoderm-like cell by culturing a human pluripotent stem cell in a culture medium comprising activin A and a GSK3β inhibitor, and a step of culturing the mesoderm-like cell in a culture medium containing BMP. The invention also provides a method for producing an isolated human PGC-like cell, which includes the aforementioned two steps and the additional step of selecting a cell positive to at least one cell surface marker selected from the group consisting of PECAM (CD31), INTEGRINa6 (CD49f), INTEGRINβ3 (CD61), KIT (CD117), EpCAM, PODOPLANIN and TRA1-81.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional of co-pending U.S. patentapplication Ser. No. 15/740,454, filed on Dec. 28, 2017, which is theU.S. national phase of International Patent Application No.PCT/JP2016/069360, filed Jun. 29, 2016, which claims the benefit ofJapanese Patent Application No. 2015-130501, filed on Jun. 29, 2015,which are incorporated by reference in their entireties herein.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: 25,226 bytes ASCII (Text) file named“747132SequenceListing.txt,” created Dec. 30, 2019.

TECHNICAL FIELD

The present invention relates to a method for inducing a primordial germcell-like cell (PGC-like cell) from a pluripotent stem cell via amesoderm-like cell, a reagent kit therefor, and a method for isolating acell belonging to a reproductive cell line obtained by the method from acell population thereof of the cell.

BACKGROUND ART

The germline is the basis for totipotency and persistence and diversityof a multicellular organism are imparted by transmitting geneticinformation and epigenetic information to the next generation in manymulticellular organisms. When an abnormality occurs in the germ line inwhich human primordial germ cells (PGCs) develop and form spermatozoonor ovum via a complicated and diversified developmental pathway, variousdevelopmental disorders such as genetic or acquired diseases anddevelopmental disorders, functional disorders, infertility and the likeappear in the offsprings. Therefore, understanding of the mechanism ofgerm cell formation is very important for the understanding of not onlybiology but also disease.

The mechanism of gametogenesis in mammals has been studied in mice andmany findings were obtained. They provide important information possiblyapplicable to all animals including human. However, in variouslyexisting animal species, elaborate mechanisms concerning germ celldevelopment vary markedly among species. For accurate understanding ineach species, findings in each animal species are considered to benecessary.

Information on the mechanism of germ cell development in human is verylacking. This is caused by the difficulty in obtaining experimentmaterials. As described above, since the mechanism of human germ cellformation is unknown, diagnosis/treatment of diseases caused by defectsin human germ cells has been difficult.

Breakthrough is said to occur by reconstituting the development of humangerm cells in vitro using human pluripotent stem cells (hPSCs) such ashuman embryonic stem cells (hESCs) (non-patent document 1) and humaninduced pluripotent stem cells (hiPSCs) (non-patent document 2).

Induction from pluripotent stem cells (PSCs) to mouse germline and thedevelopment thereafter have been reproduced in vitro (non-patentdocument 3 and non-patent document 4). That is, recent studies haveclarified that mouse (m) with pluripotency of ground state ESCs/iPSCs(non-patent document 5) is induced to pre-gastrulation ectoderm-likecells (EpiLCs), and successively to PGC-like cells (PGCLCs) havingepigenetic properties extremely similar to migratory PGCs and globaltranscription. Surprisingly, PGCLCs induced in this manner have highcapacity in both the formation of spermatozoon and ovum and progenygeneration, and these facts propose a conceptual framework for thereconstruction of human germ cell development in vitro and suggest thatsuch reconstruction can be realized.

However, hESCs/iPSCs are different from mESCs/iPSCs in terms of geneexpression profile, epigenetic property, cytokine dependency, anddifferentiation potency, and are considered to be in pluripotency statesimilar to mouse ectoderm stem cells (epiblast stem cells (EpiSCs))(non-patent document 6 and non-patent document 7). Such state is similarto post-gastrulation mouse epiblast, which means that capacity of germcell differentiation is limited (non-patent document 4). Therefore,whether hESCs/iPSCs are efficiently induced to the human germ cell fateis unknown; however, there are many reports teaching that randomdifferentiation of hESCs/iPSCs produces germ cell-like cells with lowefficiency (non-patent document 8).

DOCUMENT LIST Non-Patent Documents

-   non-patent document 1: Thomson J A, et al., Science. 282, 1145-1147,    1998-   non-patent document 2: Takahashi K, et al., Cell. 131, 861-872, 2007-   non-patent document 3: Hayashi K, et al., Science. 338, 971-975,    2012-   non-patent document 4: Hayashi K, et al., Cell. 146, 519-532, 2011-   non-patent document 5: Ying Q L, et al., Nature. 453, 519-523, 2008-   non-patent document 6: Brons I G, et al. Nature. 448, 191-195, 2007-   non-patent document 7: Tesar P J, et al., Nature. 448, 196-199, 2007-   non-patent document 8: Hayashi Y, et al., Fertil Steril. 97,    1250-1259, 2012

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

Therefore, an object of the present invention is to provide a method forinducing human primordial germ cell-like (PGC-like) cells from humanpluripotent stem cells, which is method having high efficiency and highreproducibility, thereby achieving functional reconstitution of germcell differentiation determination pathway from pluripotent stem cellsincluding ESC and iPSC in vitro. Another object of the present inventionis to provide a cell surface marker to identify human PGC-like cells.

Means of Solving the Problems

It was considered that differentiation of human pluripotent stem cellinto human PGC-like cell could be induced by the method described inHayashi K, et al., Cell. 146, 519-532, 2011 as in mouse. However, thepresent inventors used this method and found that many dead cells weredeveloped and the induction efficiency was markedly low.

To achieve an object of efficient induction of human PGC-like cells, thepresent inventors cultured human pluripotent stem cells in a culturemedium containing activin A and GSK3β inhibitor, induced mesoderm-likecells, and subjected the mesoderm-like cells to induction conditions formouse PGC-like cells. Surprisingly, they have found that the inductionefficiency of PGC-like cells increased and the development of dead cellscan be suppressed.

Furthermore, the present inventors have found at least one marker geneselected from the group consisting of PECAM (CD31), INTEGRINα6 (CD49f),INTEGRINβ3 (CD61), KIT (CD117), EpCAM, PODOPLANIN and TRA1-81 as a cellsurface marker for identifying human PGC-like cells. They have conductedfurther studies based on these findings and completed the presentinvention.

That is, the present invention relates to the following.

[1] A method for producing a human primordial germ cell-like (PGC-like)cell from a human pluripotent stem cell, comprising the following stepsI) and II):I) a step of producing a mesoderm-like cell by culturing a humanpluripotent stem cell in a culture medium comprising activin A and aGSK3β inhibitor,II) a step of culturing the mesoderm-like cell obtained in step I) in aculture medium containing BMP.[2] The method of [1] wherein the culture in the aforementioned step I)is performed for less than 60 hr.[3] The method of [2] wherein the culture in the aforementioned step I)is performed for 42 hr.[4] The method of [1] wherein the aforementioned GSK3β inhibitor isCHIR99021.[5] The method of any one of [1] to [4] wherein the culture medium inthe aforementioned step I) further comprises a fibroblast growth factorreceptor (FGFR) inhibitor.[6] The method of [5] wherein the aforementioned FGFR inhibitor isPD173074.[7] The method of any one of [1] to [6] wherein the culture medium inthe aforementioned step I) does not contain bFGF and BMP.[8] The method of any one of [1] to [7] wherein the culture medium inthe aforementioned step II) further comprises at least one cytokineselected from the group consisting of SCF, EGF and LIF.[9] The method of any one of [1] to [8] wherein the aforementionedpluripotent stem cell is a pluripotent stem cell cultured underserum-free and feeder-free conditions.[10] The method of [9] wherein the culturing under the aforementionedfeeder-free conditions is culturing on laminin511 or laminin511fragment.[11] The method of any one of [1] to [10] further comprising III) a stepof selecting a cell positive to at least one cell surface markerselected from the group consisting of PECAM (CD31), INTEGRINα6 (CD49f),INTEGRINβ3 (CD61), KIT (CD117), EpCAM, PODOPLANIN and TRA1-81 from thecells obtained in the aforementioned step II).[12] The method of [11] wherein the aforementioned step III) is a stepof selecting a double positive cell of INTEGRINα6 (CD49f) and EpCAM.[13] The method of any one of [1] to [12] wherein the aforementionedhuman pluripotent stem cell is a human iPS cell.[14] A cell population comprising a human primordial germ cell-like(PGC-like) cell produced by the method of any one of [1] to [13].[15] A method for sorting a human primordial germ cell-like (PGC-like)cell comprising selecting a cell positive to at least one cell surfacemarker selected from the group consisting of PECAM (CD31), INTEGRINα6(CD49f), INTEGRINβ3 (CD61), KIT (CD117), EpCAM, PODOPLANIN and TRA1-81.[16] The method of [15] wherein the aforementioned selection isperformed by selecting a double positive cell of INTEGRINα6 (CD49f) andEpCAM.[17] A reagent kit for inducing differentiation of a human pluripotentstem cell into a human primordial germ cell-like (PGC-like) cellcomprising the following (1) and (2):(1) a reagent for inducing a human pluripotent stem cell into amesoderm-like cell comprising activin A and a GSK3β inhibitor,(2) a reagent for inducing a mesoderm-like cell into a human primordialgerm cell-like (PGC-like) cell comprising BMP.[18] The kit of [17] wherein the aforementioned GSK3β inhibitor isCHIR99021.[19] The kit of [17] or [18] wherein the induction reagent of theaforementioned (1) further comprises a fibroblast growth factor receptor(FGFR) inhibitor.[20] The kit of [19] wherein the aforementioned FGFR inhibitor isPD173074.[21] The kit of any one of [17] to [20] wherein the induction reagent ofthe aforementioned (2) further comprises at least one cytokine selectedfrom the group consisting of SCF, EGF and LIF.[22] The kit of any one of [17] to [21] further comprising (3) a reagentfor isolating a human primordial germ cell-like (PGC-like) cellcomprising an antibody to at least one cell surface marker selected fromthe group consisting of PECAM (CD31), INTEGRINα6 (CD49f), INTEGRINβ3(CD61), KIT (CD117), EpCAM, PODOPLANIN and TRA1-81.[23] The kit of [22] wherein the isolation reagent of the aforementioned(3) comprises an antibody to INTEGRINα6 (CD49f) and an antibody toEpCAM.

Effect of the Invention

When human pluripotent stem cells are cultured in a culture mediumcontaining activin A and a GSK3β inhibitor to induce mesoderm-likecells, and the cells are cultured under conditions for induction intomouse PGC-like cells, the induction efficiency of human PGC-like cellsincreases and the development of dead cells can be suppressed. Thus,human PGC-like cells can be induced from human pluripotent stem cellswith high efficiency and good reproducibility. In addition, using thecell surface marker identified by the present invention as an index,human PGC-like cells can be efficiently isolated and purified from acell population.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of direct induction of BTAG(+) cells fromhiPSCs. View A shows bright field images of BTAG 585B1-868 hiPSCs (leftview), and the results of FACS measurement of expression of OCT3/4,SOX2, NANOG, TRA-1-60 and SSEA-4 (right view). View B is an outlinefigure showing the scheme for directly inducing BTAG(+) cells fromhiPSCs. View C shows bright field image (BF) and fluorescence images (AGand BT) of floating aggregates of hiPSCs stimulated by BMP4, SCF, EGFand LIF (left view), and bright field image (BF) and fluorescence images(AG and BT) of floating aggregates of hiPSCs under conditions withoutaddition of cytokine (right view). View D shows the results of FACSanalysis of the expression of BTAG up to day 8 during direct inductionfrom hiPSCs by BMP4, SCF, EGF and LIF (left view), and the results ofFACS analysis of the expression of BTAG up to day 8 of hiPSCs underconditions without addition of cytokine (right view). In the figures,the numbers show content percentage of BTAG(+) cells. View E shows theresults the number of BTAG(+) cells plotted for each aggregate. Theaverage is shown with the horizontal line, percentile from 25 to 75 areshown in box, and the maximum value and minimum value of the two resultsare shown with error bars. View F shows the results of gene expressionduring induction of BTAG(+) cells (hiPSCs, 2 days of induction (day2)and 6 days of induction (day6)) measured by Q-PCR. The expression levelof each gene is shown by OCT from average CT value of two housekeepinggenes of Arbp (attachment region binding protein) and Ppia(peptidylprolyl Isomerase A).

FIG. 2 shows the results of induction of BTAG(+) cells from hiPSCs viamesoderm-like cell (iMeLCs). View A is an outline figure showing thescheme for inducing BTAG(+) cells via iMeLCs. View B shows phasecontrast microscopic images of hiPSCs (left view) and iMeLCs (rightview). View C shows the results of expression of OCT3/4, SOX2 and NANOGin iMeLCs on day 42 of induction measured by FACS. View D shows brightfield image (BF) and fluorescence images (AG and BT) of floatingaggregates of hiPSCs stimulated by BMP4, SCF, EGF and LIF at respectiveinduction days (day 2, day 4, day 6 and day 8) (left view), and brightfield image (BF) and fluorescence images (AG and BT) of floatingaggregates of hiPSCs under conditions without addition of cytokine(right view). View E shows coagulation of iMeLCs stimulated by BMP4,SCF, EGF and LIF (top view), and BTAG positive cells measured by FACSunder conditions without addition of cytokine (lower view). In thefigures, the numbers show content percentage of BTAG(+) cells. View Fshows content percentage of BTAG(+) cells (left view) and the resultsthe number of BTAG(+) cells plotted for each aggregate (right view). Theaverage is shown with the horizontal line, percentile from 25 to 75 areshown in box, and the maximum value and minimum value of the two resultsare shown with error bars. View G shows the results of gene expressionduring induction of BTAG(+) cells (hiPSCs, iMeLCs, 2 days of induction(day2), 4 days of induction (day4), 6 days of induction (day6), 8 daysof induction (day8)) measured by Q-PCR. The expression level of eachgene is shown by OCT from average CT value of two housekeeping genes ofArbp and Ppia. View H shows the results of FACS measurement of theexpression of OCT3/4, SOX2 and NANOG in BTAG(+) cell on induction day 4.View I shows fluorescent staining images of BLIMP1, TFAP2C, SOX2 andSOX17 in BTAG(+)PGCLCs (primordial germ cell-like cells) (middle vieweGFP) on induction day 8 (right view). In the right figure, the brokenline shows BTAG(+)PGCLCs in the middle view.

FIG. 3 shows the study results that BTAG(+) cell is hPGCLC. View A showsthe cluster analysis results of transcriptome of BTAG(+) cells (BTAG+d6)directly induced from hiPSCs, iMeLCs, BTAG(+) cells (induction day 2 (d2), induction day 4 (d 4), induction day 6 (d 6) and induction day 8 (d8) from iMeLCs) and hiPSCs. View B shows PCA analysis results duringinduction of hiPSCs, iMeLCs and BTAG(+) cells (induction day 2 (d2BTAG+) and induction day 4 (d4 BTAG+) from iMeLCs). View C shows theresults of plotting of a gene with upward change or downward changebetween cyESCs (CMK9) and cyPGCs by comparison of expression respectivecells (left view) and a graph of frequency distribution of the gene(right view). View D shows cluster analysis results during BTAG(+)positive cell induction and based on gene expression of cell types ofcyESCs (CMK9) and cyPGCs (PGCd43, PGCd50 and PGCd51). View E showsexpression change gene (DEG) between hPGCs and H9 ESCs plotted in d6BTAG(+) cells and iMeLCs (top view) and a graph of frequencydistribution of the gene (lower view). View F shows cluster analysisresults during BTAG(+) positive cell induction and based on geneexpression of ESC and PGC. View G shows scatter diagram plotting a genewith log 2 fold change between cells before induction and d4 TNAP/NANOS3(+) cells and a gene with log 2 fold change between iMeLCs and d4BTAG(+) cells (top view), and scatter diagram plotting a gene with log 2fold change between cells before induction and d4 TNAP/NANOS3 (+) cellsand a gene with log 2 fold change between iMeLCs and d4 TNAP/NANOS3 (+)cells (lower view).

FIG. 4 shows the study results of induction pathway of hPGCLCs. View Ashows the measurement results of the number of genes with an increaseand decrease in the expression between cells of human (left view) andmouse (right view) in the main stage. View B shows the results of geneontology (GO) analysis between the cells of View A. View C showsmeasurement results of the number of genes showing high expression ineach cell type between PGCLC of human (left view) and mouse (rightview). View D shows the results of gene ontology (GO) analysis betweenthe cells of View C. View E shows Venn diagram showing overlap of humanand mouse d2 PGCLC genes shown in View C. Views F and G show the resultsof plotting of the expression of human d2 PGCLC gene (View F) and moused2 PGCLC gene (View G) in each human cell (left view) and each mousecell (right view). The average is shown with the horizontal line,percentile from 25 to 75 are shown in box, and the maximum value andminimum value of the two results are shown with error bars. View H showsheat map of the expression of a gene relating to mouse primitivepluripotency and epiblast (left view) and a gene relating to mesodermand endoderm (right view) in mESCs, mEpiLCs, mEpiSCs, hiPSCs and iMeLCs.The gene expression data was obtained from microarray analysis andRNA-seq.

FIG. 5 shows the analysis results of the surface marker of hPGCLCs. ViewA shows FACS analysis results of the expression of EpCAM and INTEGRINα6in aggregates on day 6 of induction from hiPSCs via iMeLCs. View B showsthe FACS analysis results of the expression of EpCAM and INTEGRINα6 inaggregates up to induction day 8 from hiPSCs via iMeLCs (top view). Inthe figure, Full shows induction results in BMP4, LIF, SCF and EGF, Nocytokine shows induction results under conditions without addition ofcytokine. The lower view shows the measurement results of the contentpercentage of BTAG(+) cells in the gates (P3 and P5) under respectiveconditions. View C shows the FACS analysis results of the expression ofEpCAM and INTEGRINα6 in aggregates up to induction day 6 from 585A1hiPSCs (cells free of knockin with BT and AG) via hPGCLC. In the view,Full shows induction results in BMP4, LIF, SCF and EGF, No cytokineshows induction results under conditions without addition of cytokine.View D shows the cluster analysis results of transcriptome of hiPSCs,iMeLCs, BTAG(+) cells induced via iMeLCs (day 2, day 4, day 6 and day 8)and high expressing cells (day 6) of EpCAM and INTEGRINα6 induced from585A1 hiPSCs via iMeLCs. View E shows FACS analysis results of theexpression of EpCAM and INTEGRINα6 in aggregates on day 6 of inductionvia iMeLCs induced from 1383D2 (left view) and 1383D6 (right view) usingFGFRi (top figure). The middle view and lower view shows FACS analysisresults of the expression of EpCAM and INTEGRINα6 in aggregates on day 4of induction via iMeLCs (middle view) induced from 201B7 without usingFGFRi or iMeLCs (lower view) induced using FGFRi.

FIG. 6 shows the results showing importance of BLIMP1 in hPGCLCs. View Ashows construction figure of targeting vector for BLIMP1. View B showsimmunostaining figures of the expression of AG and BLIMP1 in hPGCLCsinduced from hiPSCs (BLIMP1+/+ cells (1-7, or 1-9) or BTAG−/− cells(1-7-5 or 1-9-6)) via iMeLCs. The broken line in the lower view showsthe position of AG positive cell. View C shows a bright field image (BF)and fluorescence images (AG and BT) of cell aggregates (day 2, day 4 andday 6) induced from wild-type (left view), BLIMP1+/− (middle view) orBLIMP1−/− (right view) via iMeLCs. View D shows FACS analysis results ofthe content percentage of AG positive cells in cell aggregates (day 2,day 4, day 6 and day 8) induced from wild-type (top view), BLIMP1+/−(middle view) or BLIMP1−/− (lower view) via iMeLCs, hiPSCs and iMeLCs.View E shows the number of AG positive cells in the cell aggregatesinduced from wild-type (wt), BLIMP1+/− (het) or BLIMP1−/− (ko) viaiMeLCs. View F shows Q-PCR measurement results of the expression of eachgene in AG positive cells on day 2 or day 4 of induction from wild-type(wt) or BLIMP1−/− (ko) via iMeLCs.

FIG. 7 shows the study results of the role of BLIMP1 in neurondifferentiation. View A shows cluster analysis results of thetranscriptome of hiPSCs (wild-type (wt), BLIMP1+/− (het) or BLIMP1−/−(ko)) and AG positive cells on induction day 2 and day 4 from hiPSCs.View B shows PCA analysis results in View A. View C shows the number ofgenes that showed increased or decreased expression in BLIMP1−/− hiPSCsand AG positive cells on induction day 2 and day 4 as compared towild-type. View D shows the results of gene ontology (GO) analysis shownin View C. View E shows the results of plotting of the increased gene(left view) and decreased gene (right view) in the expression in eachcell in View C. The average is shown with the horizontal line,percentile from 25 to 75 are shown in box, and the maximum value andminimum value of the two results are shown with error bars. View F showschange of gene relating to neuron differentiation from the genes thatshowed expression change in hiPSCs and d2 and d4 hPGCLCs.

FIG. 8 shows production of BTAG-knockin hiPSCs. View A showssingle-strand annealing (SSA) activity of TALEN relative to TFAP2C (leftview) or BLIMP1 (right view). View B shows schematic showing of BLIMP1and TFAP2C gene loci and schematic showing of the knockin strategy. ViewC shows PCR screening results of the homologous recombination ofBLIMP1-2A-tdTomato (top view) and TFAP2C-2A-EGFP (lower view). View Dshows karyotype analysis results of 585A1, 585B1 and BTAG 585B1-868hiPSCs. View E shows fluorescent staining images of BLIMP1-2A-tdTomato(two top views) and TFAP2C-2A-EGFP (lower view). View F shows FACSmeasurement results of the content percentage of BTAG(+) cells per cellaggregate when BMP4, LIF, SCF and EGF were used, LIF alone was used, andKSR at each concentration was used during BTAG(+) cell induction.

FIG. 9 shows analysis results of the iMeLCs induction and propertiesthereof. View A shows bright field image, fluorescence image and FACSanalysis results of BTAG(+) cells (day 4) induced via iMeLCs or directlyinduced BTAG(+) cells (day 6). View B shows FACS analysis results duringinduction of iMeLCs (top view) and cell number measurement results(lower view). View C shows Q-PCR measurement results of each gene duringinduction of iMeLCs from hiPSCs.

FIG. 10 shows study results of the induction method of iMeLC. Views A-Eshow FACS measurement results of the content percentage of BTAG(+) cellswhen the concentration of activin A (ACTA) (View A), Wnt3A (View B),CHIR99021 (View C), BMP4 (View D) or bFGF (View E) was changed duringinduction of iMeLC from hiPSCs. View F shows phase contrast microscopicimages of the cells after 42 hr from the induction of hiPSCs using ACTAand CHIR99021, ACTA alone, CHIR99021 alone or without addition thereof.View G shows bright field image, fluorescence image and measurementresults of cell number of BTAG(+) cells when a FGFR inhibitor (FGFRi)was used. View H shows Q-PCR measurement results of the expression ofgene BTAG(+) in the cells when a FGFR inhibitor (FGFRi) was used.

FIG. 11 shows study results of the signal necessary for the induction ofBTAG(+) cells. Views A-D show bright field image, fluorescence image andFACS analysis results of BTAG(+) cells when the concentration of BMP4,the addition concentration of LDN193189, the addition of BMPs (BMP2,BMP4, BMP7 and BMP8a), or the combination of BMP4, LIF, SCF and EGF waschanged. View E shows change of each gene in BTAG(+) cells up to day 12.

FIG. 12 shows comparison of cyPGCs and cyESCs, and comparison results ofmouse pluripotent stem cells and human pluripotent stem cells. View Ashows the measurement results, by the immunostaining method, of theexpression of OCT3/4, BLIMP1, TFAP2C and DDX4 in gonad of Macacafascicularis. View B shows the measurement results, by single cellQ-PCR, of the expression of PPIA, POU5F1, NANOG, PRDM1 and TFAP2C ingonad of Macaca fascicularis. View C shows a phase contrast microscopicimage of cyESCs. View D shows the measurement results, by single cellQ-PCR, of the expression of PPIA, POU5F1, NANOG, PRDM14, GATA4 and T incyESCs. View E shows PCA analysis results of cyESC and cyPGCs. View Fshows heat map of suppression of the expression of HOX gene during PGCLCinduction in human (left view) and mouse (right view). View G showscluster analysis results of the gene expression in mESCs, EpiLCs andEpiSCs. View H shows the measurement results of the expression of 4clusters of the gene in View G.

FIG. 13 shows epigenetic analysis results of hPGCLCs. View A showsstained images of H3K9me2, H3K27me3 and 5mC and cell distribution of thefluorescence intensity. View B shows the ratio of CpG methylation ofH19, MEG3, KCNQ1 and PEG10. View C shows the analysis results, theRNA-seq method, of the expression of gene in cyESCs and gonad PGCsduring hPGCLC induction.

FIG. 14 shows production results of the BLIMP1 knockout cell line. ViewA shows SSA activity of TALEN when targeting exon4 of BLIMP1. View Bshows homologous recombination of TFAP2C-2A-EGFP (top view),BLIMP1-exon4-2A-tdTomato (middle view) and PCR analysis results oftargeting vector. View C shows scatter diagram of comparison of theexpression of RNA-seq of BLIMP1+/+ (wt) cells (left view) and d2 BTAG(+)cells (middle view) and d4 BTAG(+) cells (right view) relative toBLIMP1−/− (ko) cells. View D shows variation in the expression ofepigenetic-related genes of hPGCLC induced from BLIMP1+/+ (wt) cells,BLIMP1+/− (het) cells and BLIMP1−/− (ko) cells. View E shows change ofgene relating to neuron differentiation from the genes that showedexpression change in BLIMP1+/+ (wt) or BLIMP1−/− (ko) hiPSCs and d2 andd4 hPGCLCs induced from said cells.

DESCRIPTION OF EMBODIMENTS

The present invention provides a method for producing mesoderm-likecells from human pluripotent stem cells, which is a method for culturingthe aforementioned human pluripotent stem cells in a culture mediumadded with activin A and a glycogen synthase kinase (GSK) 3β inhibitor.

The human pluripotent stem cell to be used as a starting material may beany undifferentiated cell as long as it has “self-replicationcompetence” permitting proliferation while maintaining anundifferentiated state, and “differentiation pluripotency” permittingdifferentiation into all three primary germ layers. For example, iPScell, ES cell, embryonic germ (EG) cell, embryonic carcinoma (EC) celland the like can be mentioned, with preference given to iPS cell or EScell.

(1) Production of Human Pluripotent Stem Cell (i) ES Cell

Pluripotent stem cell can be obtained by a method known per se. Forexample, as a production method of ES cells, a method includingculturing inner cell mass of mammalian blastocyst stage (Thomson J A, etal., Science. 282, 1145-1147, 1998) can be mentioned, but the method isnot limited thereto. ES cell can be obtained from given institutions anda commercially available product can also be purchased. For example,human ES cell lines H1 and H9 are available from WiCell Institute ofUniversity of Wisconsin, and KhES-1, KhES-2 and KhES-3 are availablefrom Institute for Frontier Medical Science, Kyoto University.

(ii) iPS Cell

iPS cell can be produced by transferring a nuclear reprogrammingsubstance to the somatic cell.

(A) Sources of Somatic Cells

Examples of somatic cells that can be used as a starting material forthe production of iPS cell include keratinizing epithelial cells (e.g.,keratinized epidermal cells), mucosal epithelial cells (e.g., epithelialcells of the superficial layer of tongue), exocrine gland epithelialcells (e.g., mammary gland cells), hormone-secreting cells (e.g.,adrenomedullary cells), cells for metabolism or storage (e.g., livercells), intimal epithelial cells constituting interfaces (e.g., type Ialveolar cells), intimal epithelial cells of the obturator canal (e.g.,vascular endothelial cells), cells having cilia with transportingcapability (e.g., airway epithelial cells), cells for extracellularmatrix secretion (e.g., fibroblasts), constrictive cells (e.g., smoothmuscle cells), cells of the blood and the immune system (e.g., Tlymphocytes), sense-related cells (e.g., rod cells), autonomic nervoussystem neurons (e.g., cholinergic neurons), sustentacular cells ofsensory organs and peripheral neurons (e.g., satellite cells), nervecells and glia cells of the central nervous system (e.g., astrogliacells), pigmenT cells (e.g., retinal pigment epithelial cells),progenitor cells thereof (tissue progenitor cells) and the like. Thereis no limitation on the degree of cell differentiation, and the like;even undifferentiated progenitor cells (including somatic stem cells)and finally differentiated mature cells can be used alike as sources ofsomatic cells in the present invention. Examples of undifferentiatedprogenitor cells include tissue stem cells (somatic stem cells) such asfat derived from stroma (stem) cells, neural stem cells, hematopoieticstem cells, mesenchymal stem cells, and dental pulp stem cells.

Somatic cells isolated from a mammal can be pre-cultured using a mediumknown per se suitable for their cultivation according to the choice ofcells. Examples of such media include, but are not limited to, minimalessential medium (MEM) containing about 5 to 20% fetal bovine serum(FCS), Dulbecco's modified Eagle medium (DMEM), RPMI1640 medium, 199medium, F12 medium, and the like. When a transfer reagent such ascationic liposome, for example, is used in bringing a cell into contactwith nuclear reprogramming substances and another iPS cell establishmentefficiency improver, it is sometimes preferable that the medium havebeen replaced in advance with a serum-free medium so as to prevent thetransfer efficiency from decreasing.

(b) Nuclear Reprogramming Substance

In the present invention, “a nuclear reprogramming substance” may beconfigured with any substance, such as a proteinous factor or a nucleicacid that encodes the same (including a form integrated in a vector), ora low molecular compound, as long as it is a substance (substances)capable of inducing an iPS cell from a somatic cell. When the nuclearreprogramming substance is a proteinous factor or a nucleic acid thatencodes the same, preferable nuclear reprogramming substance isexemplified by the following combinations (hereinafter, only the namesfor proteinous factors are shown).

(1) Oct3/4, Klf4, c-Myc(2) Oct3/4, Klf4, c-Myc, Sox2 (here, Sox2 is replaceable with Sox1,Sox3, Sox15, Sox17 or Sox18; Klf4 is replaceable with Klf1, Klf2 orKlf5; c-Myc is replaceable with T58A (active mutant), N-Myc or L-Myc)(3) Oct3/4, Klf4, c-Myc, Sox2, Fbx15, Nanog, Eras, ECAT15-2, TclI,β-catenin (active mutant S33Y)(4) Oct3/4, Klf4, c-Myc, Sox2, TERT, SV40 Large T antigen (hereinafterSV40 LT)(5) Oct3/4, Klf4, c-Myc, Sox2, TERT, HPV16 E6(6) Oct3/4, Klf4, c-Myc, Sox2, TERT, HPV16 E7(7) Oct3/4, Klf4, c-Myc, Sox2, TERT, HPV16 E6, HPV16 E7(8) Oct3/4, Klf4, c-Myc, Sox2, TERT, Bmi1[For further information of the above-mentioned factors, see WO2007/069666 (however, in the combination (2) above, for replacement ofSox2 with Sox18, and replacement of Klf4 with Klf1 or Klf5, see NatureBiotechnology, 26, 101-106 (2008)); for details of the combination“Oct3/4, Klf4, c-Myc, Sox2”, see also Cell, 126, 663-676 (2006), Cell,131, 861-872 (2007) and the like. For details of the combination of“Oct3/4, Klf4, c-Myc, Sox2”, see also Cell, 126, 663-676 (2006), Cell,131, 861-872 (2007) and the like. For details of the combination of“Oct3/4, Klf2 (or Klf5), c-Myc, Sox2”, see also Nat. Cell Biol., 11,197-203 (2009). For details of the combination of “Oct3/4, Klf4, c-Myc,Sox2, hTERT, SV40LT”, see also Nature, 451, 141-146 (2008).)(9) Oct3/4, Klf4, Sox2 (see also Nature Biotechnology, 26, 101-106(2008))

(10) Oct3/4, Sox2, Nanog, Lin28 (see Science, 318, 1917-1920 (2007))

(11) Oct3/4, Sox2, Nanog, Lin28, hTERT, SV40LT (see Stem Cells, 26,1998-2005 (2008))(12) Oct3/4, Klf4, c-Myc, Sox2, Nanog, Lin28 (see Cell Research (2008)600-603)(13) Oct3/4, Klf4, c-Myc, Sox2, SV40LT (see also Stem Cells, 26,1998-2005 (2008))

(14) Oct3/4, Klf4 (see Nature 454:646-650 (2008), Cell Stem Cell,2:525-528 (2008))

(15) Oct3/4, c-Myc (see Nature 454:646-650 (2008))

(16) Oct3/4, Sox2 (see Nature, 451, 141-146 (2008), WO 2008/118820) (17)Oct3/4, Sox2, Nanog (see WO 2008/118820) (18) Oct3/4, Sox2, Lin28 (seeWO 2008/118820)

(19) Oct3/4, Sox2, c-Myc, Esrrb (Esrrb is replaceable with Esrrg. SeeNat. Cell Biol., 11, 197-203 (2009))(20) Oct3/4, Sox2, Esrrb (see Nat. Cell Biol., 11, 197-203 (2009))

(21) Oct3/4, Klf4, L-Myc (22) Oct3/4, Nanog (23) Oct3/4

(24) Oct3/4, Klf4, c-Myc, Sox2, Nanog, Lin28, SV40LT (see Science,324:797-801 (2009))

In the above-mentioned (1)-(24), a member of other Oct family, forexample, Oct1A, Oct6 and the like can also be used instead of Oct3/4. Inaddition, a member of other Sox family, for example, Sox7 and the likecan also be used instead of Sox2 (or Sox1, Sox3, Sox15, Sox17, Sox18).Furthermore, a member of other Lin family, for example, Lin28b and thelike can also be used instead of Lin28.

Any combination that does not fall in (1) to (24) above but comprisesall the constituents of any one of (1) to (22) and further comprises anoptionally chosen other substance can also be included in the scope of“nuclear reprogramming substance” in the present invention. Providedthat the somatic cell to undergo nuclear reprogramming is endogenouslyexpressing one or more of the constituents of any one of (1) to (24)above at a level sufficient to cause nuclear reprogramming, acombination of only the remaining constituents excluding the one or moreconstituents can also be included in the scope of “nuclear reprogrammingsubstance” in the present invention.

Of these combinations, at least one, preferably two or more, morepreferably three or more selected from Oct3/4, Sox2, Klf4, c-Myc, Nanog,Lin28 and SV40LT are preferable nuclear reprogramming substances.

Among these combinations, when the obtained iPS cell is to be used fortherapeutic purposes, a combination of 3 factors of Oct3/4, Sox2 andKlf4 (i.e., the above-mentioned (9)) is preferable. On the other hand,when the iPS cell is not to be used for therapeutic purposes (e.g., usedas an investigational tool for drug discovery screening and the like), 4factors of Oct3/4, Sox2, Klf4 and c-Myc, 5 factors of Oct3/4, Klf4,c-Myc, Sox2 and Lin28, or 6 factors further including Nanog (i.e., theabove-mentioned (12)) or 7 factors further including SV40 Large T (i.e.,the above-mentioned (24)), is preferable.

Furthermore, the above-mentioned combination with L-Myc instead of c-Mycis also a preferable example of a nuclear reprogramming substance.

Information on the mouse and human cDNA sequences of the aforementionednuclear reprogramming substances is available with reference to the NCBIaccession numbers mentioned in WO 2007/069666 (in the publication, Nanogis described as ECAT4. Mouse and human cDNA sequence information onLin28, Lin28b, Esrrb, Esrrg and L-Myc can be acquired by referring tothe following NCBI accession numbers, respectively); those skilled inthe art are easily able to isolate these cDNAs.

Name of gene Mouse Human Lin28 NM_145833 NM_024674 Lin28b NM_001031772NM_001004317 Esrrb NM_011934 NM_004452 Esrrg NM_011935 NM_001438 L-MycNM_008506 NM_001033081

When a proteinous factor is used as a nuclear reprogramming substance,it can be prepared by inserting the cDNA obtained into an appropriateexpression vector, transferring it into a host cell, culturing the cell,and recovering the recombinant proteinous factor from the cultured cellsor a conditioned medium therefor. Meanwhile, when a nucleic acid thatencodes a proteinous factor is used as a nuclear reprogrammingsubstance, the cDNA obtained is inserted into a viral vector, plasmidvector, episomal vector or the like to construct an expression vector,which is subjected to the nuclear reprogramming step.

(c) Method of Introducing Nuclear Reprogramming Substance into SomaticCell

Introduction of the nuclear reprogramming substance with a somatic cell,when the substance is a proteinaceous factor, can be achieved using amethod known per se for protein transfer into a cell. In considerationof clinical application to human, iPS cell to be the starting materialtherefore is also preferably produced without gene manipulation.

Such methods include, for example, the method using a protein transferreagent, the method using a protein transfer domain (PTD)- or cellpenetrating peptide (CPP)-fusion protein, the microinjection method andthe like. Protein transfer reagents are commercially available,including those based on a cationic lipid, such as BioPOTER ProteinDelivery Reagent (Gene Therapy Systems), Pro-Ject™ Protein TransfectionReagent (PIERCE) and ProVectin (IMGENEX); those based on a lipid, suchas Profect-1 (Targeting Systems); those based on a membrane-permeablepeptide, such as Penetrain Peptide (Q biogene) and Chariot Kit (ActiveMotif), GenomONE (ISHIHARA SANGYO KAISHA, LTD.) utilizing HVJ envelope(inactive hemagglutinating virus of Japan) and the like. The transfercan be achieved per the protocols attached to these reagents, a commonprocedure being as described below. The nuclear reprogramming substanceis diluted in an appropriate solvent (e.g., a buffer solution such asPBS or HEPES), a transfer reagent is added, the mixture is incubated atroom temperature for about 5 to for 15 minutes to form a complex, thiscomplex is added to cells after exchanging the medium with a serum-freemedium, and the cells are incubated at 37° C. for one to several hours.Thereafter, the medium is removed and replaced with a serum-containingmedium.

Developed PTDs include those using transcellular domains of proteinssuch as drosophila-derived AntP, HIV-derived TAT (Frankel, A. et al,Cell 55, 1189-93 (1988) or Green, M. & Loewenstein, P. M. Cell 55,1179-88 (1988)), Penetratin (Derossi, D. et al, J. Biol. Chem. 269,10444-50 (1994)), Buforin II (Park, C. B. et al. Proc. Natl Acad. Sci.USA 97, 8245-50 (2000)), Transportan (Pooga, M. et al. FASEB J. 12,67-77 (1998)), MAP (model amphipathic peptide) (Oehlke, J. et al.Biochim. Biophys. Acta. 1414, 127-39 (1998)), K-FGF (Lin, Y. Z. et al.J. Biol. Chem. 270, 14255-14258 (1995)), Ku70 (Sawada, M. et al. NatureCell Biol. 5, 352-7 (2003)), Prion (Lundberg, P. et al. Biochem.Biophys. Res. Commun. 299, 85-90 (2002)), pVEC (Elmquist, A. et al. Exp.Cell Res. 269, 237-44 (2001)), Pep-1 (Morris, M. C. et al. NatureBiotechnol. 19, 1173-6 (2001)), Pep-7 (Gao, C. et al. Bioorg. Med. Chem.10, 4057-65 (2002)), SynBl (Rousselle, C. et al. Mol. Pharmacol. 57,679-86 (2000)), HN-I (Hong, F. D. & Clayman, G L. Cancer Res. 60, 6551-6(2000)), and HSV-derived VP22. CPPs derived from the PTDs includepolyarginines such as 11R (Cell Stem Cell, 4,381-384 (2009)) and 9R(Cell Stem Cell, 4, 472-476 (2009)).

A fused protein expression vector incorporating cDNA of a nuclearreprogramming substances and PTD or CPP sequence is prepared, andrecombination expression is performed using the vector. The fusedprotein is recovered and used for transfer. Transfer can be performed inthe same manner as above except that a protein transfer reagent is notadded.

Microinjection, a method of placing a protein solution in a glass needlehaving a tip diameter of about 1 μm, and injecting the solution into acell, ensures the transfer of the protein into the cell.

When the establishment efficiency of iPS cells is important, the nuclearreprogramming substance is also preferably used in the form of a nucleicacid encoding a proteinaceous factor rather than the proteinaceousfactor itself. The nucleic acid may be a DNA or an RNA, or a DNA/RNAchimera. The nucleic acid may be double-stranded or single-stranded.Preferably, the nucleic acid is a double-stranded DNA, particularlycDNA.

cDNA of a nuclear reprogramming substance is inserted into anappropriate expression vector comprising a promoter capable offunctioning in a host somatic cell. Useful expression vectors include,for example, viral vectors such as retrovirus, lentivirus, adenovirus,adeno-associated virus, herpes virus and Sendai virus, plasmids for theexpression in animal cells (e.g., pA1-11, pXT1, pRc/CMV, pRc/RSV,pcDNAI/Neo) and the like.

The type of a vector to be used can be chosen as appropriate accordingto the intended use of the iPS cell to be obtained. Useful vectorsinclude adenoviral vector, plasmid vector, adeno-associated viralvector, retroviral vector, lentiviral vector, Sendai viral vector,episomal vector and the like.

Examples of promoters used in expression vectors include the EF1αpromoter, the CAG promoter, the SRα promoter, the SV40 promoter, the LTRpromoter, the CMV (cytomegalovirus) promoter, the RSV (Rous sarcomavirus) promoter, the MoMuLV (Moloney mouse leukemia virus) LTR, theHSV-TK (herpes simplex virus thymidine kinase) promoter and the like,with preference given to the EF1α promoter, the CAG promoter, the MoMuLVLTR, the CMV promoter, the SRα promoter and the like.

The expression vector may contain as desired, in addition to a promoter,an enhancer, a polyadenylation signal, a selectable marker gene, a SV40replication origin and the like. Examples of selectable marker genesinclude the dihydrofolate reductase gene, the neomycin resistant gene,the puromycin resistant gene and the like.

Nucleic acid as a nuclear reprogramming substance (reprogramming gene)may be incorporated on individual expression vectors, 2 or more kinds,preferably 2-3 kinds, of genes may be incorporated into one expressionvector. The former case is preferable when using a retroviral orlentiviral vector that offers high gene transfer efficiency, and thelatter is preferable when using a plasmid, adenoviral, or episomalvector and the like. Furthermore, an expression vector incorporating 2or more kinds of genes, and other expression vector incorporating onegene alone can also be used in combination.

In the context above, when multiple reprogramming genes are integratedin one expression vector, these genes can preferably be integrated intothe expression vector via a sequence enabling polycistronic expression.By using a sequence enabling polycistronic expression, it is possible tomore efficiently express a plurality of genes integrated in oneexpression vector. Useful sequences enabling polycistronic expressioninclude, for example, the 2A sequence of foot-and-mouth disease virus(PLoS ONE 3, e2532, 2008, Stem Cells 25, 1707, 2007), the IRES sequence(U.S. Pat. No. 4,937,190) and the like, with preference given to the 2Asequence.

An expression vector harboring a nucleic acid which is a nuclearreprogramming substance can be introduced into a cell by a techniqueknown per se according to the choice of the vector. In the case of aviral vector, for example, a plasmid containing the nucleic acid isintroduced into an appropriate packaging cell (e.g., Plat-E cells) or acomplementary cell line (e.g., 293-cells), the viral vector produced inthe culture supernatant is recovered, and the vector is infected to acell by a method suitable for the viral vector. For example, specificmeans using a retroviral vector are disclosed in WO2007/69666, Cell,126, 663-676 (2006) and Cell, 131, 861-872 (2007). Specific means usinga lentiviral vector is disclosed in Science, 318, 1917-1920 (2007). WhenPGC-like cell induced from iPS cell is utilized as a regenerativemedicine for infertility treatment, gene therapy of germ cell and thelike, since expression (reactivation) of reprogramming gene may increasethe carcinogenic risk of germ cell or reproductive tissue regeneratedfrom PGC-like cell derived from iPS cell, nucleic acid encoding anuclear reprogramming substance is preferably expressed transiently,without being integrated into the chromosome of the cells. From thisviewpoint, it is preferable to use an adenoviral vector, which isunlikely to be integrated into the chromosome, is preferred. Specificmeans using an adenoviral vector is disclosed in Science, 322, 945-949(2008). Adeno-associated virus vector is unlikely to be integrated intothe chromosome, and is less cytotoxic and less phlogogenic thanadenoviral vectors, so that it is another preferred vector. Sendai virusvectors are capable of being stably present outside of the chromosome,and can be degraded and removed using an siRNA as required, so that theyare preferably utilized as well. Useful Sendai virus vectors aredescribed in J. Biol. Chem., 282, 27383-27391 (2007) or JP-B-3602058.

When a retroviral vector or a lentiviral vector is used, even ifsilencing of the transgene has occurred, it possibly becomes reactive;therefore, for example, a method can be used preferably wherein anucleic acid encoding nuclear reprogramming substance is cut out usingthe Cre-loxP system, when becoming unnecessary. That is, with loxPsequences arranged on both ends of the nucleic acid in advance, iPScells are induced, thereafter the Cre recombinase is allowed to act onthe cells using a plasmid vector or adenoviral vector, and the regionsandwiched by the loxP sequences can be cut out. Because theenhancer-promoter sequence of the LTR U3 region possibly upregulates ahost gene in the vicinity thereof by insertion mutation, it is morepreferable to avoid the expression regulation of the endogenous gene bythe LTR outside of the loxP sequence remaining in the genome withoutbeing cut out, using a 3′-self-inactive (SIN) LTR prepared by deletingthe sequence, or substituting the sequence with a polyadenylationsequence such as of SV40. Specific means using the Cre-loxP system andSIN LTR is disclosed in Chang et al., Stem Cells, 27: 1042-1049 (2009).

Meanwhile, being a non-viral vector, a plasmid vector can be transferredinto a cell using the lipofection method, liposome method,electroporation method, calcium phosphate co-precipitation method, DEAEdextran method, microinjection method, gene gun method and the like.Specific means using a plasmid as a vector are described in, forexample, Science, 322, 949-953 (2008) and the like.

When a plasmid vector, an adenovirus vector and the like are used, thetransfection can be performed once or more optionally chosen times(e.g., once to 10 times, once to 5 times or the like). When two or morekinds of expression vectors are introduced into a somatic cell, it ispreferable that these all kinds of expression vectors be concurrentlyintroduced into a somatic cell; however, even in this case, thetransfection can be performed once or more optionally chosen times(e.g., once to 10 times, once to 5 times or the like), preferably thetransfection can be repeatedly performed twice or more (e.g., 3 times or4 times).

Also when an adenovirus or a plasmid is used, the transgene can getintegrated into chromosome; therefore, it is eventually necessary toconfirm the absence of insertion of the gene into chromosome by Southernblotting or PCR. For this reason, like the aforementioned Cre-loxPsystem, it can be advantageous to use a means wherein the transgene isintegrated into chromosome, thereafter the gene is removed. In anotherpreferred mode of embodiment, a method can be used wherein the transgeneis integrated into chromosome using a transposon, thereafter atransposase is allowed to act on the cell using a plasmid vector oradenoviral vector so as to completely eliminate the transgene from thechromosome. As examples of preferable transposons, piggyBac, atransposon derived from a lepidopterous insect, and the like can bementioned. Specific means using the piggyBac transposon is disclosed inKaji, K. et al., Nature, 458: 771-775 (2009), Woltjen et al., Nature,458: 766-770 (2009).

Another preferable non-integration type vector is an episomal vector,which is capable of self-replication outside of the chromosome. Specificmeans using an episomal vector is disclosed by Yu et al., in Science,324, 797-801 (2009). Where necessary, an expression vector may beconstructed by inserting a reprogramming gene into an episomal vectorhaving loxP sequences placed in the same orientation on the 5′ and 3′sides of a vector component essential for the replication of theepisomal vector, and transferred to a somatic cell.

Examples of the episomal vector include a vector comprising as a vectorcomponent a sequence derived from EBV, SV40 and the like necessary forself-replication. The vector component necessary for self-replication isspecifically exemplified by a replication origin and a gene that encodesa protein that binds to the replication origin to control thereplication; examples include the replication origin oriP and the EBNA-1gene for EBV, and the replication origin ori and the SV40 large Tantigen gene for SV40.

The episomal expression vector comprises a promoter that controls thetranscription of a reprogramming gene. The promoter used may be asdescribed above. The episomal expression vector may further contain asdesired an enhancer, a polyadenylation signal, a selection marker geneand the like, as described above. Examples of the selection marker geneinclude the dihydrofolate reductase gene, the neomycin resistance geneand the like.

An episomal vector can be transferred into a cell using, for example,the lipofection method, liposome method, electroporation method, calciumphosphate co-precipitation method, DEAE dextran method, microinjectionmethod, gene gun method and the like. Specifically, for example, methodsdescribed in Science, 324: 797-801 (2009) and elsewhere can be used.

Whether or not the vector component necessary for the replication of thereprogramming gene has been removed from the iPS cell can be confirmedby performing a Southern blot analysis or PCR analysis using a part ofthe vector as a probe or primer, with the episome fraction isolated fromthe iPS cell as a template, and determining the presence or absence of aband or the length of the band detected. The episome fraction can beprepared by a method obvious in the art; for example, methods describedin Science, 324: 797-801 (2009) can be used.

When the nuclear reprogramming substance is a low-molecular-weightcompound, the substance can be introduced into a somatic cell bydissolving the substance at a suitable concentration in an aqueous ornon-aqueous solvent, adding the solution to a medium suitable for theculture of somatic cell isolated from human or mouse (e.g., minimumessential medium (MEM), Dulbecco's modified Eagle medium (DMEM),RPMI1640 medium, 199 medium, F12 medium and the like containing about5-20% fetal bovine serum such that the concentration of a nuclearreprogramming substance is sufficient to cause nuclear reprogramming inthe somatic cell and free of cytotoxicity, and culturing the cells for agiven period. While the concentration of the nuclear reprogrammingsubstance varies depending on the kind of the nuclear reprogrammingsubstance to be used, it is appropriately selected from the range ofabout 0.1 nM-about 100 nM. The contact period is not particularlylimited as long as it is sufficient for achieving nuclear reprogrammingof the cell. Generally, they may be co-existed in the medium untilpositive colony emerges.

(d) Establishment Efficiency Improving Substance for iPS Cell

Since the iPS cell establishment efficiency has been low, varioussubstances that improve the efficiency have recently been proposed oneafter another. It can be expected, therefore, that the iPS cellestablishment efficiency will be increased by bringing anotherestablishment efficiency improver, in addition to the aforementionednuclear reprogramming substance, into contact with the transfer subjectsomatic cell.

Examples of the iPS cell establishment efficiency improving substanceinclude, but are not limited to, histone deacetylase (HDAC) inhibitors[e.g., low-molecular inhibitors such as valproic acid (VPA) (Nat.Biotechnol., 26(7):795-797 (2008), trichostatin A, sodium butyrate, MC1293, and M344, nucleic acid-based expression inhibitors such as siRNAsand shRNAs against HDAC (e.g., HDAC1 siRNA Smartpool® (Millipore), HuSH29 mer shRNA Constructs against HDAC1 (OriGene) and the like), and thelike], DNA methyl transferase inhibitors (e.g., 5-azacytidine) (Nat.Biotechnol., 26(7):795-797 (2008)), G9a histone methyl transferaseinhibitors [for example, low-molecular inhibitors such as BIX-01294(Cell Stem Cell, 2:525-528 (2008)), and nucleic acid-based expressioninhibitors such as siRNAs and shRNAs (Cell Stem Cell, 3, 475-479 (2008))against G9a], L-channel calcium agonist (e.g., Bayk8644) (Cell StemCell, 3, 568-574 (2008)), p53 inhibitor (e.g., siRNA and shRNA to p53,UTF1 (Cell Stem Cell, 3, 475-479 (2008)), Wnt Signaling (e.g., solubleWnt3a) (Cell Stem Cell, 3, 132-135 (2008)), 2i/LIF (2i is inhibitor ofmitogen-activated protein kinase signalling and glycogen synthasekinase-3, PloS Biology, 6(10), 2237-2247 (2008)) and the like. Thenucleic acid-based expression inhibitors mentioned above may be in theform of expression vectors harboring a DNA that encodes an siRNA orshRNA.

Of the aforementioned constituents of nuclear reprogramming substances,SV40 large T, for example, can also be included in the scope of iPS cellestablishment efficiency improvers because it is an auxiliary factorunessential for the nuclear reprogramming of somatic cells. While themechanism of nuclear reprogramming remains unclear, it does not matterwhether auxiliary factors, other than the factors essential for nuclearreprogramming, are deemed nuclear reprogramming substances or iPS cellestablishment efficiency improvers. Hence, because the somatic cellnuclear reprogramming process is taken as an overall event resultingfrom contact of a nuclear reprogramming substance and an iPS cellestablishment efficiency improver with a somatic cell, it does notalways seems to be essential for those skilled in the art to distinguishbetween the two.

An iPS cell establishment efficiency improver can be contacted with asomatic cell as mentioned above for each of (a) when the substance is aproteinous factor and (b) when the substance is a nucleic acid encodingthe proteinous factor, or (c) when the substance is alow-molecular-weight compound.

An iPS cell establishment efficiency improver may be contacted with asomatic cell simultaneously with a nuclear reprogramming substance, andeither one may be contacted in advance, as far as the iPS cellestablishment efficiency from a somatic cell improves significantlycompared with the efficiency obtained in the absence of the improver. Inan embodiment, for example, when the nuclear reprogramming substance isa nucleic acid that encodes a proteinous factor and the iPS cellestablishment efficiency improver is a chemical inhibitor, the iPS cellestablishment efficiency improver can be added to the medium after thecell is cultured for a given length of time after the gene transfertreatment, because the nuclear reprogramming substance involves a givenlength of time lag from the gene transfer treatment to themass-expression of the proteinous factor, whereas the iPS cellestablishment efficiency improver is capable of rapidly acting on thecell. In another embodiment, for example, when the nuclear reprogrammingsubstance and iPS cell establishment efficiency improver are both usedin the form of a viral vector or plasmid vector, both may besimultaneously transferred into the cell.

(e) Improving the Establishment Efficiency by Culture Conditions

The iPS cell establishment efficiency can further be improved byculturing the cells under hypoxic conditions in the nuclearreprogramming process for somatic cells. As mentioned herein, the term“hypoxic conditions” means that the ambient oxygen concentration as ofthe time of cell culture is significantly lower than that in theatmosphere. Specifically, conditions involving lower oxygenconcentrations than the ambient oxygen concentrations in the 5-10%CO₂/95-90% air atmosphere, which is commonly used for ordinary cellculture, can be mentioned; examples include conditions involving anambient oxygen concentration of 18% or less. Preferably, the ambientoxygen concentration is 15% or less (e.g., 14% or less, 13% or less, 12%or less, 11% or less and the like), 10% or less (e.g., 9% or less, 8% orless, 7% or less, 6% or less and the like), or 5% or less (e.g., 4% orless, 3% or less, 2% or less and the like). The ambient oxygenconcentration is preferably 0.1% or more (e.g., 0.2% or more, 0.3% ormore, 0.4% or more and the like), 0.5% or more (e.g., 0.6% or more, 0.7%or more, 0.8% or more, 0.95% or more and the like), or 1% or more (e.g.,1.1% or more, 1.2% or more, 1.3% or more, 1.4% or more and the like).

While any method of creating a hypoxic state in a cellular environmentcan be used, the easiest way is to culture cells in a CO₂ incubatorpermitting adjustments of oxygen concentration, and this represents asuitable case. CO₂ incubators permitting adjustment of oxygenconcentration are commercially available from various manufacturers(e.g., CO₂ incubators for hypoxic culture manufactured by Thermoscientific, Ikemoto Scientific Technology, Juji Field, Wakenyaku etc.).

The time of starting cell culture under hypoxic conditions is notparticularly limited, as far as iPS cell establishment efficiency is notprevented from being improved compared with the normal oxygenconcentration (20%). The start time may be before or after the somaticcell is contacted with the nuclear reprogramming substance, or at thesame time as the contact, or after the contact, it is preferable, forexample, that the culture under hypoxic conditions be started just afterthe somatic cell is contacted with the nuclear reprogramming substance,or at a given time interval after the contact [e.g., 1 to 10 (e.g., 2,3, 4, 5, 6, 7, 8 or 9) days].

The duration of cultivation of cells under hypoxic conditions is notparticularly limited, as far as iPS cell establishment efficiency is notprevented from being improved compared with the normal oxygenconcentration (20%); examples include, but are not limited to, periodsof 3 days or more, 5 days or more, for 7 days or more or 10 days ormore, and 50 days or less, 40 days or less, 35 days or less or 30 daysor less and the like. Preferred duration of cultivation under hypoxicconditions varies depending on ambient oxygen concentration; thoseskilled in the art can adjust as appropriate the duration of cultivationaccording to the oxygen concentration used. In an embodiment of thepresent invention, if iPS cell candidate colonies are selected with drugresistance as an index, it is preferable that a normal oxygenconcentration be restored from hypoxic conditions before starting drugselection.

Furthermore, preferred starting time and preferred duration ofcultivation for cell culture under hypoxic conditions also varydepending on the choice of nuclear reprogramming substance used, iPScell establishment efficiency at normal oxygen concentrations and thelike.

After contacting a nuclear reprogramming substance (and iPS cellestablishment efficiency improving substance), for example, 10-15%FBS-containing DMEM, DMEM/F12 or DME culture medium (these culture mediacan further contain LIF, penicillin/streptomycin, puromycin,L-glutamine, nonessential amino acids, β-mercaptoethanol and the like asappropriate) or a commercially available culture medium [for example,culture medium for primate ES cell (culture medium for primate ES/iPScell, Reprocell), serum-free medium (mTeSR, Stemcell Technologies)] andthe like.

Examples of the culture method include contacting a somatic cell with areprogramming factor on 10% FBS-containing DMEM or DMEM/F12 culturemedium at 37° C. in the presence of 5% CO₂ and culturing for about 4-7days, thereafter reseeding the cells on feeder cells (e.g., mitomycinC-treated STO cells, SNL cells etc.), and culturing the cells in abFGF-containing culture medium for primate ES cell from about 10 daysafter the contact of the somatic cell and the reprogramming factor,whereby iPS-like colonies can be obtained after about 30-about 45 daysor longer from the contact.

Alternatively, the cells are cultured on feeder cells (e.g., mitomycinC-treated STO cells, SNL cells etc.) at 37° C. in the presence of 5% CO₂in a 10% FBS-containing DMEM culture medium (which can further containLIF, penicillin/streptomycin, puromycin, L-glutamine, nonessential aminoacids, β-mercaptoethanol and the like as appropriate), whereby ES-likecolonies can be obtained after about 25-about 30 days or longer.Desirably, a method using a somatic cell itself to be reprogrammed,instead of the feeder cells (Takahashi K, et al. (2009), PLoS One.4:e8067 or WO2010/137746), or an extracellular substrate (e.g.,Laminin-5 (WO2009/123349) and Matrigel (BD)).

A candidate colony of iPS cells can be selected in two ways: methodswith drug resistance and reporter activity as indicators, and methodsbased on macroscopic examination of morphology. As an example of theformer, a colony positive for drug resistance and/or reporter activityis selected using a recombinant cell wherein the locus of a gene highlyexpressed specifically in pluripotenT cells (e.g., Fbx15, Nanog, Oct3/4and the like, preferably Nanog or Oct3/4) is targeted by a drugresistance gene and/or a reporter gene. Examples of such recombinan Tcells include MEFs derived from a mouse having the βgeo (which encodes afusion protein of β-galactosidase and neomycin phosphotransferase) geneknocked in to the Fbx15 gene locus [Takahashi & Yamanaka, Cell, 126,663-676 (2006)], and MEFs derived from a transgenic mouse having thegreen fluorescent protein (GFP) gene and the puromycin resistance geneintegrated in the Nanog gene locus [Okita et al., Nature, 448, 313-317(2007)]. On the other hand, methods for selecting a candidate colony bymacroscopic examination of morphology include, for example, the methoddescribed by Takahashi et al. in Cell, 131, 861-872 (2007). Although themethods using reporter cells are convenient and efficient, colonyselection by macroscopic examination is desirable from the viewpoint ofsafety when iPS cells are prepared for therapeutic purposes in humans.When 3 factors of Oct3/4, Klf4 and Sox2 are used as the nuclearreprogramming substance, the number of the established clones decreases,but almost all resulting colonies are iPS cells having high qualitycomparable to that of ES cell. Therefore, iPS cell can be establishedefficiently even without using a reporter cell.

The identity of the cells of the selected colony as iPS cells can beconfirmed by positive responses to Nanog (or Oct3/4) reporters(puromycin resistance, GFP positivity and the like), as well as by thevisible formation of an ES cell-like colony, as described above;however, to ensure greater accuracy, it is possible to perform testssuch as analyzing the expression of various ES-cell-specific genes, andtransplanting the selected cells to a mouse and confirming teratomaformation.

The human pluripotent stem cells obtained by the aforementioned methodand cultured using feeder cells and serum show diversity, and therefore,are desirably cultured under restricted culture conditions. Suchserum-free and feeder-free conditions include, for example, the methoddescribed in Nakagawa M, et al., Sci Rep. 4, 3594, 2014 and the like,and a method including culturing on an extracellular substrate (e.g.,laminin5 (WO 2009/123349), laminin5 fragment (e.g., Laminin-5E8 (Nippi.Inc.)) and Matrigel (BD)) in a serum-free medium (e.g., mTeSR (StemcellTechnology), Essential 8 (Life Technologies) and StemFit (Ajinomoto Co.,Inc.)).

(2) Differentiation Induction from Human Pluripotent Stem Cell intoMesoderm-Like Cell (Step I))

Examples of the basic medium for differentiation induction which is usedin step I) include, but are not limited to, Neurobasal medium, NeuralProgenitor Basal medium, NS-A medium, BME medium, BGJb medium, CMRL 1066medium, minimum essential medium (MEM), Eagle MEM medium, aMEM medium,Dulbecco's modified Eagle medium (DMEM), Glasgow MEM medium, ImprovedMEM Zinc Option medium, IMDM medium, Medium 199 medium, DMEM/F12 medium,ham medium, RPMI 1640 medium, Fischer's medium, and a mixed medium ofthese and the like.

The medium may be a serum-containing medium or serum-free medium.Preferably, a serum-free medium is used. The serum-free medium (SFM)means a medium free of an untreated or unpurified serum, and therefore,a medium containing purified blood-derived component or animaltissue-derived component (growth factor and the like) can be mentioned.The concentration of the serum (e.g., fetal bovine serum (FBS), humanserum and the like) may be 0-20%, preferably 0-5%, more preferably 0-2%,most preferably 0% (that is, serum-free). SFM may or may not contain anoptional serum replacement. Examples of the serum replacement includealbumin (e.g., lipid-rich albumin, albumin substitute recombinantalbumin and the like, plant starch, dextran and protein hydrolysateetc.), transferrin (or other iron transporter), fatty acid, insulin,collagen precursor, trace element, 2-mercaptoethanol, 3′-thioglycerol ora substance containing an equivalent of these and the like asappropriate. Such serum replacement can be prepared, for example, by themethod described in WO 98/30679. To simplify more, a commerciallyavailable product can be utilized. Examples of such commerciallyavailable substance include Knockout (trade mark) Serum Replacement(KSR), Chemically-defined Lipid concentrated, and Glutamax(Invitorogen).

The medium may contain other additives known per se. The additive is notparticularly limited as long as mesoderm-like cell equivalent toepiblast cell before intestinal invagination is produced by the methodof the present invention. For example, growth factors (e.g., insulin andthe like), polyamines (e.g., putrescine and the like), minerals (e.g.,sodium selenite and the like), saccharides (e.g., glucose and the like),organic acids (e.g., pyruvic acid, lactic acid and the like), aminoacids (e.g., non-essential amino acid (NEAA), L-glutamine and the like),reducing agents (e.g., 2-mercaptoethanol and the like), vitamins (e.g.,ascorbic acid, d-biotin and the like), steroids (e.g., [beta]-estradiol,progesterone and the like), antibiotics (e.g., streptomycin, penicillin,gentamicin and the like), buffering agents (e.g., HEPES and the like),nutrition additives (e.g., B27 supplement, N2 supplement,StemPro-Nutrient Supplement and the like) can be mentioned. Eachadditive is preferably contained in a concentration range known per se.

The medium for differentiation induction of human pluripotent stem cellsinto mesoderm-like cells contains a basal medium and activin A and aGSK-3β inhibitor as essential additives.

The concentration of activin A in the medium for differentiationinduction is, for example, not less than about 5 ng/ml, preferably notless than about 10 ng/ml, more preferably not less than about 15 ng/mland, for example, not more than about 40 ng/ml, preferably not more thanabout 30 ng/ml, more preferably not more than 25 ng/ml.

In the present invention, the GSK-3β inhibitor is defined as a substancethat inhibits kinase activity of GSK-3β protein (e.g., phosphorylationcapacity against β catenin), and many are already known. Examplesthereof include lithium chloride (LiCl) first found as a GSK-3βinhibitor, BIO, which is an indirubin derivative (alias, GSK-3βinhibitor IX; 6-bromo indirubin 3′-oxime), SB216763 which is a maleimidederivative(3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione),GSK-3β inhibitor VII which is a phenyl α-bromomethyl ketone compound(4-dibromoacetophenone), L803-mts which is a cell membrane-permeabletype-phosphorylated peptide (alias, GSK-3β peptide inhibitor;Myr-N-GKEAPPAPPQSpP-NH₂) and CHIR99021 having high selectivity(6-[2-[4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino]ethylamino]pyridine-3-carbonitrile).These compounds are commercially available from, for example,Calbiochem, Biomol and the like, and can be easily utilized. They may beobtained from other sources, or may be directly produced.

The GSK-3β inhibitor used in step I) can preferably be CHIR99021.

The concentration of CHIR99021 in the medium is, though not particularlylimited to, for example, 0.1 μM-50 μM is preferable, for example, 1 μM,2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM or a concentrationnot less than these. Preferably, a concentration higher than theconcentration 1 μM of a drug generally used for inhibition of GSK-3β isused, and it is more preferably not less than 3 μM.

In consideration of the subsequent induction step (step II)) intoPGC-like cell, the medium is preferably free of basic fibroblast growthfactor (bFGF) and bone morphogenic protein (BMP).

The medium preferably further contains a fibroblast growth factorreceptor (FGFR) inhibitor.

In the present invention, the FGFR inhibitor is not particularly limitedas long as it is a drug inhibiting binding of FGF receptor and FGF orsignal transduction occurring after the binding. For example, PD173074or BGJ398 can be mentioned.

When PD173074 is used, the concentration in the medium is notparticularly limited. It is preferably 1 nM-50 nM, for example, 1 nM, 2nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 25 nM, 30 nM, 35nM, 40 nM, 45 nM, 50 nM, more preferably 25 nM.

The medium preferably further contains KSR. KSR present in an effectiveconcentration range remarkably increases induction efficiency ofmesoderm-like cell. The concentration of KSR is, for example, 5%, 10%,15%, 20% or above, more preferably 15%.

In culturing here, the medium preferably further contains a ROCKinhibitor to suppress apoptosis during separation of human pluripotentstem cells into single cells. The ROCK inhibitor is not particularlylimited as long as it can suppress function of the Rho kinase (ROCK).For example, Y-27632 may be preferably used in the present invention.

The concentration of Y-27632 in the medium is, though not particularlylimited to, preferably 1 μM-50 μM, for example, 1 μM, 2 μM, 3 μM, 4 μM,5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM,16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50μM, more preferably 10 μM.

An incubator used for inducing pluripotent stem cells into mesoderm-likecells is not particularly limited, and flask, tissue culture flask,dish, petri dish, tissue culture dish, multidish, microplate, microwellplate, multiplate, mutiwell plate, microslide, chamber slide, petridish, tube, tray, culture bag, and roller bottle can be mentioned. Theincubator may be cell adhesive. A cell adhesive incubator may be coatedwith any cell adhesion substrate such as extracellular matrix (ECM) andthe like for the purpose of improving adhesiveness of the incubatorsurface to the cells. The cell adhesion substrate may be any substanceaiming at adhesion of pluripotent stem cells or feeder cells (whenused). As the cell adhesion substrate, collagen, gelatin, poly-L-lysine,poly-D-lysine, poly-L-ornithine, laminin, and fibronectin and a mixturethereof, such as Matrigel and lysed cellular membrane preparations canbe mentioned. More preferred is an incubator coated with fibronectin.

For culturing, human pluripotent stem cells are seeded on theabove-mentioned incubator to a cell density of, for example, about10⁴-10⁵ cells/cm², preferably about 2-6×10⁴ cells/cm², and culturedunder an atmosphere of 1-10% CO₂/99-90% air in an incubator at about30-40° C., preferably about 37° C., for less than 60 hr, preferably 42hr (e.g., error of ±2 hr is tolerable).

The fact of differentiation into mesoderm-like cells can be confirmed,for example, by analyzing the expression level of the marker gene ofmesoderm-like cell and/or pluripotent stem cell by RT-PCR. Mesoderm-likecells are defined as cells having either or both of the followingproperties:

(1) an increase in at least one gene expression selected from T, EOMES(Eomesodermin), EVX1 (Even-Skipped Homeobox 1), SP5 (Sp5 transcriptionfactor), MIXL1 (Mix Paired-Like Homeobox 1) and NODAL, compared topluripotent stem cell before differentiation induction,(2) a decrease in at least one gene expression selected from POU5F1 (POUdomain, class 5, transcription factor 1), NANOG and SOX2 (SRY (SexDetermining Region Y)—Box 2), compared to pluripotent stem cell beforedifferentiation induction.

As mentioned above, the medium for differentiation induction intomesoderm-like cell contains activin A and a GSK3β inhibitor. Therefore,the present invention also provides a reagent kit for differentiationinduction of pluripotent stem cells into mesoderm-like cells, whichcontains activin A and a GSK3β inhibitor. These components may beprovided in the form of being dissolved in water or a suitable buffer,provided as a freeze-dry powder and can also be used upon dissolution ina suitable solvent when in use. Also, these components may be placed ina kit each as a single reagent, or two or more kinds thereof may bemixed and provided as a single reagent as long as they do not adverselyinfluence each other.

(3) Differentiation Induction of Mesoderm-Like Cell into Human PGC-LikeCell (Step II))

Differentiation of the thus-obtained mesoderm-like cells into PGC-likecells can be induced by culturing in the presence of BMP. Therefore, asecond aspect of the present invention relates to a method for producinghuman PGC-like cells from human pluripotent stem cells via mesoderm-likecells obtained by the method of the above-mentioned (2). Therefore, themethod for inducing differentiation of human pluripotent stem cells intohuman PGC-like cells of the present invention includes

I) a step of producing mesoderm-like cells from human pluripotent stemcells according to any method described in the above-mentioned (2); andII) a step of culturing the mesoderm-like cells obtained in step I) inthe presence of BMP.

The basal medium for differentiation induction of human PGC-like cellsin step II), the basal medium exemplified to be used in step I) ispreferably used in the same manner.

The medium may be a serum-containing medium or serum-free medium (SFM).Preferably, a serum-free medium is used. The medium preferably furthercontains KSR. KSR present in an effective concentration range remarkablyincreases induction efficiency of mesoderm-like cell. The concentrationof KSR is, for example, 5%, 10%, 15%, 20% or above, more preferably 15%.

BMP used as an essential additive for the medium for differentiationinduction of mesoderm-like cells into PGC-like cells is BMP2, BMP4 orBMP7. A more preferable BMP is BMP 2 or BMP 4. The concentration of BMPis, for example, not less than about 100 ng/ml, not less than about 200ng/ml, not less than about 300 ng/ml, not less than about 400 ng/ml.

The medium for differentiation induction into PGC-like cells preferablyfurther contains at least one cytokine selected from the groupconsisting of stem cell factor (SCF), epithelial cell growth factor(EGF) and leukemia inhibitory factor (LIF) as an additive.

The concentration of SCF is, for example, not less than about 50 ng/ml,not less than about 100 ng/ml, not less than about 200 ng/ml, not lessthan about 300 ng/ml, more preferably 100 ng/ml.

The concentration of LIF is, for example, not less than about 300 U/ml,not less than about 500 U/ml, not less than about 800 U/ml, or not lessthan about 1000 U/ml, more preferably 1,000 U/ml.

The concentration of SCF is, for example, not less than about 30 ng/ml,not less than about 50 ng/ml, not less than about 80 ng/ml, not lessthan about 100 ng/ml, more preferably 100 ng/ml.

The concentration of EGF is, for example, not less than about 10 ng/ml,not less than about 20 ng/ml, not less than about 30 ng/ml, not lessthan about 40 ng/ml, not less than about 50 ng/ml, more preferably 50ng/ml.

In culturing in step II), the medium preferably further contains a ROCKinhibitor to suppress apoptosis during separation of mesoderm-like cellsinto single cells. As the ROCK inhibitor, one which is the same as theabove is used.

For culturing, mesoderm-like cells are seeded in a cell non-adhesive orlow adhesive incubator known per se to a cell density of, for example,about 1-50×10³ cells/cm², preferably about 5-20×10³ cells/cm², andcultured under an atmosphere of 1-10% CO₂/99-90% air in an incubator atabout 30-40° C., preferably about 37° C., for about 4-10 days,preferably 4-8 days, more preferably about 6 days (e.g., 144±12 hr,preferably 144±6 hr).

The fact of differentiation into PGC-like cells can be confirmed, forexample, by analyzing the expression of BLIMP1 by RT-PCR and the like.Where necessary, expression of other gene and cellular surface antigencan also be examined. As other gene, TFAP2C can be mentioned. Whenpluripotent stem cells having a fluorescence protein gene under controlof BLIMP1- and/or TFAP2C-promoter is used as a starting material, thefact of differentiation into PGC-like cells can be confirmed by FACSanalysis. When pluripotent stem cells derived from human or othernon-mouse mammal such as ESC or iPSC and the like do not have anappropriate transgenic reporter, the fact of differentiation intoPGC-like cells is preferable confirmed by FACS analysis and the likeusing one or more kinds of cellular surface antigens specificallyexpressed in PGC-like cells. Examples of the cellular surface antigeninclude at least one marker gene selected from the group consisting ofPECAM (CD31), INTEGRINα6 (CD49f), INTEGRINβ3 (CD61), KIT (CD117), EpCAM,PODOPLANIN and TRA1-81, with preference given to INTEGRINα6 (CD49f) andEpCAM.

To isolate PGC-like cell, it is preferable to further perform, as stepIII), a step of selecting a cell positive to the aforementioned cellularsurface antigen from the cells obtained in the aforementioned step II).

Isolation of PGC-like cell can be performed by a method known per se.For example, isolation of PGC-like cell may be performed by cell sortingusing an antibody to the cellular surface antigen described above.

As mentioned above, in a preferable embodiment, the medium fordifferentiation induction of mesoderm-like cells into PGC-like cellscontains BMP. Therefore, the present invention also provides a reagentkit containing BMP for differentiation induction of mesoderm-like cellsinto PGC-like cells. These components may be provided in the form ofbeing dissolved in water or a suitable buffer, provided as a freeze-drypowder and can also be used upon dissolution in a suitable solvent whenin use. Also, these components may be placed in a kit each as a singlereagent, or two or more kinds thereof may be mixed and provided as asingle reagent as long as they do not adversely influence each other.

Assuming isolation of PGC-like cells, the reagent kit fordifferentiation induction may contain an antibody to the aforementionedcellular surface antigen.

(4) Cell Population Containing Pluripotent Stem Cell-Derived PGC-LikeCells Via Mesoderm-Like Cell

The present invention also provides a cell population containingpluripotent stem cell-derived PGC-like cells, which is produced by theaforementioned steps I) and II). The cell population is preferably apurified population of PGC-like cells, and preferably a cell populationpurified by the aforementioned step III).

While the present invention is more specifically explained by referringto the following Examples, it is needless to say that the presentinvention is not limited thereby.

EXAMPLES Experiment Method Ethical Guidelines

All animal experiments were performed according to the ethicalguidelines of Kyoto University and Siga University of Medical Science.The induction experiments from hiPSCs into hPGCLCs were approved by theKyoto University Institutional Review Board (Institutional EthicsCommittee).

Culture of hiPSCs

hiPSC strains (201B7, 585A1, 585B1) (Takahashi K, et al., Cell. 131,861-872, 2007 and Okita K, et al., Stem Cells. 31, 458-466, 2013) weresubjected to maintenance culture under mitomycin C (MMC)-treated SNLfeeder cell by a conventional method [Dulbecco's Modified Eagle Medium(DMEM/F12; Life Technologies) added with 20% (v/v) Knockout SerumReplacement (KSR; Life Technologies), 1% GlutaMax (Life Technologies),0.1 mM non-essential amino acid, 4 ng/ml recombinant human bFGF (Wako),and 0.1 mM 2-mercaptoethanol], and then adapted to feeder-freeconditions [on recombinant laminin511 (rLN511E8) (iMatrix-511,Nippi)-coated cell culture plate, StemFit™ (Ajinomoto, Tokyo, Japan)medium] (Nakagawa M, et al., Sci Rep. 4, 3594, 2014). To separate thecells to single cells for passage culture and differentiation induction,the cells were treated with a 1:1 mixed solution of TrypLE Select (LifeTechnologies) and 0.5 mM EDTA/PBS, and 10 μM ROCK inhibitor (Y-27632;Wako Pure Chemical Industries) was added for 24 hr after seeding.

Production of BTAG Knockin Reporter Strain

For construction of a donor vector for isolating BLIMP1-p2A-tdTomato(hereinafter BT) or TFAP2C-p2A-eGFP (hereinafter AG) knockin hiPSCstrains, the homology arm of BLIMP1 [left(5-prime)arm: 1148 bp; right(3-prime)arm: 1192 bp] and the homology arm of TFAP2C [left arm: 1144bp; right arm: 1162 bp] were amplified by PCR and subcloned to pCR2.1vector (TOPO TA Cloning; Life Technologies). p2A-tdTomato fragment orp2A-eGFP fragment respectively having PGK-Neo cassette or PGK-Purocassette each having adjacent loxP moiety was amplified by PCR and,using GeneArt Seamless Cloning & Assembly Kit (Life Technologies),inserted into the 3′-terminal of BLIMP1 coding sequence or TFAP2C codingsequence of the above-mentioned subclone vector containing a homologyarm. Then, MC1-DT-A-polyA cassette was introduced into the downstream ofthe right (3′) homology arm of BLIMP1-p2A-tdTomato donor vector orTFAP2C-p2A-eGFP donor vector by restriction enzyme NotI/XbaI orSacI/KpnI.

TALEN sequence targeting a sequence adjacent to the stop codon of BLIMP1and TFAP2C was produced using GoldenGate TALEN and TAL Effector kit(Addgene, #1000000016). The RVD sequences of TALEN are described below;

BLIMP1-left (5-prime), NN NN NG NI HD HD NG NN NG NI NI NI NN NN NG HDNI NI NI;BLIMP1-right (3-prime), HD NG NG NI NI NN NN NI NG HD HD NI NG NG NN NNNG NG HD;

TFAP2C-left, NN NN NI NN NI NI NT HD NI HD NI NN NN NI NI NI NG;TFAP2C-right, NI HD NG HD NG HD HD NG NI NI HD HD NG NG NG HD NG.

The TALEN activity was evaluated by SSA assay (FIG. 8, View A) (SakumaT, et al., Genes Cells. 18, 315-326, 2013). As the control in thisevaluation, pGL4-SSA empty reporter plasmid, pGL4-SSA-HPRT1 reporterplasmid, and TALEN pair targeting human HPRT1 were used.

Using NEPA21 type II (Nepa gene), BLIMP1-p2A-tdTomato donor vector orTFAP2C-p2A-eGFP donor vector (5 μg) and TALEN plasmid (2.5 μg each) wereintroduced into hiPSCs (585A1 or 585B1) by electroporation. About 12days later, a single colony was isolated, and whether it was targetingintegration or random integration was evaluated by PCR of extractiongenome DNA. To exclude indel mutation, untargeted allele (Non-targetedalleles) was further screened by the Sanger sequence method. To removePGK-Neo cassette and PGK-Puro cassette, a plasmid expressing Crerecombinase was transfected into a strain having an insertion targetingboth BLIMP1 and TFAP2C gene locus.

Differentiation Induction into Endoderm or Trophectoderm

Aiming at endoderm differentiation, hiPSCs having BLIMP1-p2A-tdTomatoknockin gene locus or TFAP2C-p2A-eGFPknockin gene locus were seeded iniMatrix-511 coating plate and cultured in RPMI1640 (Wako Pure ChemicalIndustries) containing 2% B27 (Life Technologies), 100 ng/ml activin A(Peprotech, #120-14), and 3 μM CHIR99021 (Biovision, #1677-5). Aiming atdifferentiation into trophectoderm, iPSCs were seeded in iMatrix-511(Nippi, 892001)-coating plate and cultured in bFGF-free StemFit™(HumanZyme, # HZ-1078) containing 10 ng/ml BMP4.

Production of BLIMP1-Knockin/Knockout hiPSCs

For construction of donor vector isolating BLIMP1-p2A-tdTomato;BLIMP1^(−/−) knockin/knockout hiPSC strain, a homology arm adjacentBLIMP1 exon 4 [left (5-prime)arm: 1517 bp; right (3-prime)arm: 1446 bp]was amplified by PCR, and subcloned to pCR2.1 vector. 2A-tdTomato-SV40polyA fragment having PGK-Neo cassette adjacent to LoxP site wasamplified by PCR. Then, using GeneArt Seamless Cloning & Assembly Kit,additional 30 bp sequence of intron 4 was inserted instead of exon4analogue. Next, MC1-DT-A-polyA cassette was introduced into thedownstream of right (3′-terminal) homology arm by using restrictionenzymes XhoI and SacI.

TALEN construct having the RVD sequence shown below and targeting BLIMP1exon 4 was produced by the aforementioned method;

left (5-prime), HD NI HD HD NI HD NG NG HD NI NG NG NN NI HD NN NN HD;right (3-prime), NI NN HD NN HD NI NG HD HD NI NN NG NG NN HD NG NG NG.

The TALEN activity was evaluated by SSA assay (FIG. 14, View A). Forisolation of BTAG; BLIMP1^(+/−) and BTAG; BLIMP1^(−/−) hiPSCs strains,the aforementioned donor vector (5 μg) and TALEN plasmid (each 2.5 μg)were introduced into TFAP2C-p2A-eGFP (AG) knockin hiPSC strain. Thesuccess of random targeting unaccompanied by integration was evaluatedby PCR of extracted genome DNA. Untargeted allele was further evaluatedby Sanger sequence method, assuming presence (BTAG; BLIMP1^(−/−)) orabsence (BTAG; BLIMP1^(+/−)) of frameshift indel mutation.

Karyotype Classification and Gband Analysis

hiPSCs were incubated in 100 ng/ml demecolcine-containing medium for 8hr. After separation with Accutase (Sigma-Aldrich), and the cells weretreated with hypotonic buffer (Genial Genetics, GGS-JL006b) warmed inadvance and incubated at 37° C. for 30 min. Then, the cells were fixedwith Carnoy's solution (3:1 mixture of methanol and acetic acid), andadded dropwise on a glass slide on paper sheet immersed in water.Chromosomes fluorescenced by DAPI staining were counted to determinekaryotype. G band analysis was performed by Nihon Gene ResearchLaboratoryies Inc. (Sendai, Japan).

Induction of iMeLCs and hPGCLCs

Initial mesoderm-like cells (iMeLCs) were induced by seeding hiPSCsmaintained in StemFit™ in a 12 well plate coated with human plasmafibronectin (Millipore, FC010) at 1.0-2.0×10⁵ cells per well in GK15medium [GMEM containing 15% KSR, 0.1 mM NEAA, 2 mM L-glutamine, 1 mMsodium pyruvate and 0.1 mM 2-mercaptoethanol (Life Technologies)]containing 50 ng/ml activin A, 3 μM CHIR99021 and 10 μM ROCK inhibitor(Y-27632; Wako Pure Chemical Industries). The hiPSCs were induced byseeding in a low-cell-binding V-bottom 96 wellplate (Thermo, 81100574)at 3.0×10³ cells per well in GK15 added with 1000 U/ml L1F (Millipore, #LIF1005), 200 ng/ml BMP 4,100 ng/ml SCF (R&D Systems, 455-MC), 50 ng/mlEGF (R&D Systems, 236-EG), and 10 μM ROCK inhibitor. For investigationof conditions and induction of iMeLCs or hPGCLC, PD173074 (StemGent,#04-0008) or LDN193189 (StemGent, #04-0074) was respectively added.Other than the above, WNT3A (R&D Systems, 5036-WN) was used instead ofCHIR99021, and BMP2 (R&D Systems, 355-BM), BMP7 (R&D Systems, 354-BP),or BMP8A (R&D Systems, 1073-BP) was used instead of BMP4.

FACS Analysis

Floating aggregates containing hPGCLCs were separated by treating with0.05% Trypsin-EDTA/PBS at 37° C. for 10 min. After washing with PBScontaining FBS and 0.1% BSA, the cell suspension was filtered by a cellstrainer (BD Biosciences) and centrifuged to remove cell aggregates. Thecollected cells were suspended in FACS buffer (0.1% BSA in PBS) andanalyzed by a flow cytometer (ARIA III; BD Biosciences). For theanalysis of hPGCLCs or hiPSCs, the separated cells were stained withAPC-conjugated anti-human CD326 (EpCAM), BV421-conjugatedanti-human/mouse CD49f, PE-conjugated anti-TRA-1-60, or FITC-conjugatedanti-SSEA-4. Furthermore, intracellular staining using Alexa fluor647-conjugated anti-OCT3/4, Alexa Fluor 647-conjugated anti-NANOG orV450-conjugated anti-SOX2 was performed using BD cytofix/CytofermFixation/Permeabilization kit (BD, 554714) and according to theManufacturer's instructions. The antibodies used are shown in Table 1.

TABLE 1 Antibody Supplier Catalogue# APC-conjugated anti- BioLegend324207 CD326 Alexa Fluor 647- BD Pharmingen 560329 conjugatedanti-OCT3/4 Alexa Fluor 647- BD Pharmingen 561300 conjugated anti-NANOGBV421-conjugated anti- BioLegend 313623 CD49f FITC-conjugated anti- BDPharmingen 560126 SSEA-4 PE-conjugated anti-TRA- BD Pharmingen 5601931-60 V450-conjugated anti- BD Horizon 561610 SOX2Preparation of Single Cell cDNA from cyESCs and cyPGCs

cyESCs (CMK9) (Macaca fascicularis ES cell) were obtained from Dr.Suemori (Fujioka T, et al., Int J Dev Biol. 48, 1149-1154, 2004 and;Suemori H, et al., Dev Dyn. 222, 273-279, 2001) and cultured togetherwith mouse fetal feeder cells (mouse embryonic feeders (MEFs)) in hESCmedium [DMEM/FI2 (Life Technologies) added with 20% (vol/vol) KSR (LifeTechnologies), 1 mM sodium pyruvate (Life Technologies, 2 mM GlutaMax(Life Technologies), 0.1 mM non-essential amino acid (LifeTechnologies), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich), 1000 U/mL ESGROmouse LIF (Millipore), 4 ng/ml recombinant human bFGF (Wako PureChemical Industries)] in a conventional method. For SC3-seq analysis(Nakamura T, et al., Nucleic Acids Res. 43, e60. 2015), the cells weretreated with CTK solution [0.25% trypsin (Life Technologies), 0.1 mg/mLof collagenase IV (Life Technologies), 1 mM CaCl₂ (Nacalai Tesque)],incubate at 37° C. for about 10 min and in 0.25% trypsin/PBS(Sigma-Aldrich) at 37° C. for about 10 min. Thereafter, the cells weredispersed in 1% (vol/vol) KSR/PBS to give single cells.

In Macaca fascicularis, ovumrecovery, intracytoplasm spermatozooninjection (ICSI), preimplantation embryo culture, and transplantation ofpreimplantation embryo to individual were performed according toconventional methods (Yamasaki J, et al., Theriogenology. 76, 33-38,2011). The transferred embryos were monitored by ultrasonicationdiagnosis, and removed by caesarean section on embryonic days 43, 50 and51. The gender of the embryo was determined by sex specific PCR ofgenome DNA isolated from the somatic cell tissue (Wilson and Erlandsson,Biol Chem. 379, 1287-1288, 1998). The genital ridge was incised,separated into single cells in 0.25% trypsin/PBS at 37° C. for about 10min, and pipetting was repeated. The obtained single cells weredispersed in 0.1 mg/ml PVA/PBS (Sigma-Aldrich) and subjected to SC3-seqanalysis.

Q-PCR and RNA-Seq Analysis

RNA extraction for PCR was performed using RNeasy Micro Kit (QIAGEN) andaccording to the Manufacturer's instructions. cDNA synthesis andamplification using 1 ng of purified total RNA, and construction of cDNAlibrary for RNA sequence were performed according to the methoddescribed in Nakamura T, et al., Nucleic Acids Res. 43, e60. 2015. Q-PCRwas performed by measuring Power SYBR Green PCR Master mix (LifeTechnologies) along with amplified cDNA by using CFX384 real-time qPCRsystem (Bio-Rad). The gene expression level was evaluated by calculatingΔC_(t) (log 2 scale) normalized to average ΔC_(t) values of PPIA andARBP. The primers used are shown in Table 2 (human gene) and Table 3(Macaca fascicularis gene).

TABLE 2 Gene Forward primer Reverse primer BLIMP1 AAAGCAAAGCATCACGTTGACAGGATGGATGGTGAGAGAAGCAA TFAP2C ATTAAGAGGATGCTGGGGTCTGCACTGTACTGCACACTCACCTT NANOS3 TGGCAAGGGAAGAGCTGAAATCTTATTGAGGGCTGACTGGATGC DAZL TGGCCCTTCTTTCAGTGACTTC GACCCTAGGGGGCACTAGTAADPPA3 AAGCCCAAAGTCAGTGAGATGA GCTATAGCCCAACTACCTAATGC DDX4TTCTTCACAAGCTGCCAATCCA TTCTTCTCTGCATCAAAACCACA ZFP42CCAGACTGGATAACAGCAAGAGC TGCAAATTTTTCATTCTCTAGGGC PRDM14TATCATACTGTGCACTTGGCAGAA AGCAACTGGGACTACAGGTTTGT KLF2ACTAGAGGATCGAGGCTTGTGA TGCCCACCTGTCTCTCTATGTA KLF4AGCCTAAATGATGGTGCTTGGT CCTTGTCAAAGTATGCAGCAGT TCL1BCAAATCCCCTTCATACCCACCA TGCCATCTCTTAAACCGAACCA TFCP2L1AGCTCAAAGTTGTCCTACTGCC TTCTAACCCAAGCACAGATGGC ESRRBTAAAATGGCAGTTCGCCATTGC CCAGATAGATGGGACCAGGATG POU5F1CTGTCTCCGTCACCACTGTG AAACCCTGGCACAAACTCCA NANOG AGAGGTCTCGTATTTGOTGCATAAACACTCGGTGAAATCAGGGT SOX2 TGAATCAGTCTGCCGAGAATCCTCTCAAACTGTGCATAATGGAGT SOX15 TTTTAATCCAGCAGCATCCGCTAATTGTATGTTGTGCGGCTCTC SOX17 TTCGTGTGCAAGCCTGAGAT TAATATACCGCGGAGCTGGCGATA4 CGTCTTTCTCAGCAGAGCTGTA CTCTGGTACAGCCAGTAGGATT GATA6ACAGGGCGATTTGCTTTCAGTT CTTCTGTTGGGGGTAACGTCTG FOXA2ACCCGGTTTTATCCCTTGAATC ATACAACCTGCAACCAGACAGG MIXL1TGCTTTCAAAACACTCGAGGAC GAGTGATCGAAGTAACAGGTGC SP5 GAGATTTGAAACAGTGCTCGGGGGAGCTGAAGACAAAAGCAACA EOMES AAGGGGAGAGTTTCATCATCCCGGCGCAAGAAGAGGATGAAATAG NODAL CATTGCCTCAGGCTGGGTTGGTACAGCTCATTAGCAGAGAACCA T AGCCAAAGACAATCAGCAGAAA CACAAAAGGAGGGGGTTCACTAEVX1 GAAATCCTCACTCCCACACTCA GAAGAAGCACTCCGTCTCAGTC GSCGTCGAGAAAGAGGAACGAGGAG AAATACTAGGGTGGGGGGTAGT MSX2GGCAGAAGGTAAAGCCATGTTT TAAAGGTATACCGGAGGGAGGG PAX6GCGGGTGACAAAATAGTTGTCTT GCCAGGATGTCAAATCTCTCCA SOX1GGCCAAGGTAACACTCATCGTA ACCCTGTGATTTGGGAAGTGAA DNMT3ATGGGATTCATGCAGACTCATGC AAAGTGAGAAACTGGGCCTGAA DNMT3BTAACTGGAGCCACGAGGTAAC GCATCCGTCATCTTTCAGCCTA DNMT3L AGCCATAAGGAGCAGGCACTGGGGAGAAAGCAGTTCTTCACCA HOXD1 AGCTGCTTCAGTGATCTTCACAACCCATTCTGTGGATTTGTTCA PPIA TTGATCATTTGGTGTGTTGGGCAAGACTGAGATGCACAAGTGGT ARBP GAAACTCTGCATTCTCGCTTCCACTCGTTTGTACCCGTTGATGA ERCC 1806 GATCCCGGAAGATACGCTCTAAGCGCAGGTTGATGCTTCCAATAAA ERCC 451.5 CAGGCAAGAGTTCAATCGCTTAGTAGCCCTTCAGTGACTGTGAGATG ERCO 56.4 CCAACCCCACATTGTAACTTCGGTCTTTACTTACGCGCTCCTCT

TABLE 3 Gene Forward primer Reverse primer BLIMP1TTCCCAACTACTCGTTTGTTCTTTG CATGTAAGAGGCAGAAAAAGGAAGG TFAP2CTCGGAGATCAAGTCCTCTGG CCTTTGAACACGGGGTTTAG PRDM14 TGCCCTGTTGTTTTAGGACTGTAACCAGCAGTTAAGGAAAGGCT POU5F1 GGGAGGAGCTAGGGAAAGAGAACCTACCCCCACCCGTTGTGTTCCCA NANOG TGTTCCGGTTTCCATTATGCC TAGGCTCCAACCATACTCCAGATA4 CAAATCCCCTTCATACCCACCA TGCCATCTCTTAAACCGAACCA TTGCTGTCCCAAGTGGCTTAC CTGGACCCTGGCAAACATCT

Reading of Mapping of RNA-Seq and Conversion to Gene Expression Level

Genome sequences [mouse GRCm38/mm10, human GRCh37/hg19, and Macacafascicularis MacFas5.0] and transcript annotation (mouse ref GRCm38,human ref_GRCh37, and Macaca fascicularis ref MacFas5.0) were obtainedfrom NCBI ftp site (ftp://ftp-trace.ncbi.nlm.nih.gov/genomes/Macacafascicularis).

Read trimming, mapping and expression level were evaluated according tothe method described above (Nakamura T, et al., Nucleic Acids Res. 43,e60. 2015). Library adapter and poly-A sequence were eliminated by cutadapt-1.3. The reads smaller than 30 bp were eliminated. All remainingreads were mapped on the genome by using “no-coverage-search” option(Kim D, et al., Genome Biol. 14, R36, 2013) and utilizing ERCC spike-inRNAs (Life Technologies) accompanying top hat-1.4.1/bowtie1.0.1. Thereads mapped on the genome from ERCC spike-in RNAs using Perl scriptwere separated, and cufflinks-2.2.0 program (Trapnell C et al., NatBiotechnol. 28, 511-515, 2010) was performed on ref MacFas5.0transcription annotation by using “compatible-hits-norm”,“no-length-correction” and “library-type fr-secondstrand” options. Allreference copies were extended by 10 Kb from the transcriptiontermination site (TTSs) to correct insufficient annotation data(Nakamura T, et al., Nucleic Acids Res. 43, e60. 2015). All reads mappedon the ERCC spike-in RNA sequence were used for evaluation of thetranscription copy number per cell. The expression level was normalizedonly for all mapping reads (RPM), and further analyzed by log₂ (RPM+1).

Comparison of Gene Expression in Human, Macaca fascicularis and Mouse

For comparison of Macaca fascicularis gene and human gene, a one-to-onecorrespondence table of genes was made by genome genomic coordinatecomparison. First, all human transcription annotations (ref_GRCh37containing all exon data) were converted to genome gene loci of Macacafascicularis by using LiftOver utility(https://genome.ucsc.edu/cgi-bin/hgLiftOver). The chain files used forLiftOver (hg19ToMacFas5.over.chain and macFas5ToHg19.over.chain) wereobtained from http://hgdownload-test.cse.ucsc.edu/goldenPath/. Then,human genome annotation at MacFas5.0 gene loci was compared with refMacFas5.0, and searches for gene locusting MacFas5.0 transcriptionproduct were successively performed. The same method for ref MacFas5.0was performed, the transcription annotation at ref MacFas5.0 wasconverted to hg19 gene loci by using LiftOver, and searches for genelocusting human transcription product were performed. The whole 17,932genes were identified by two kinds of comparison to find a gene thatdefinitely matches between human (24,968) and Macaca fascicularis gene(29,437). In human and mouse genes (ref GRCm38,26,556 genes), the samecomparison was performed, and 15,941 genes matched. Since KLF2 gene wasnot annotated by ref MacFas5.0, annotation of this gene was added to refMacFas5.0 reference gff file in the corresponding region of human KLF2.

Transcriptome Analysis

To analyze specifically expressing genes, UHC analysis (R3.1.1 Euclideandistances and hclust function accompanying Ward distance function) andPCA analysis (R3.1.1 prcomp function) were used. In this case, genesshowing maximum log₂ (RPM+1) value of less than 4 were excluded. Thegenes (DEGs) specifically expressed in hPGCLC induction were selectedbased on P value of one-way Anova test (calculated by value function offalse positive rate <0.01, R3.1.1) and fold changes of two sequentialtime point (>2). DEGs in BLIMP1^(−/−) cell as compared with wild-typesample were selected based on the P value (<0.05) of the student t-testand fold change (>2). GO analysis was performed using DAVID web tool(Huang da W, et al., Nat Protoc. 4, 44-57, 2009). A heatmap was madeusing R3.1.1, gplotspackage, heatmap.2 function. For comparison ofRNA-seq data and GSE30056 data (Affymetrix GeneChip Mouse Genome 430 2.0Array), RNA-seq data of mESCs after normalization using the standardcurve of the mESCs array data was calculated.

Immunofluorescence Analysis

For immunofluorescent staining of hPGCLCs, BTAG positive cells in thecell aggregates on day 8 of induction from BTAG 585B1-868 hiPSCs viaiMeLC were selected by FACS, mixed with BTAG 585B1-868 hiPSCs at 1:1ratio and spread on MAS-coated slide glass (Matsunami). The slide wasfixed with 4% para-formaldehyde (PFA) for 15 min, and washed 3 timeswith PBS and once with PBST. After a permeation treatment with PBScontaining 0.5% triton X at room temperature for 5 min, the slide waswashed 3 times with PBS. Then, the slide was incubated in a blockingsolution (5% normal goat serum, 0.2% tween 20, 1×PBS) at roomtemperature for 2 hr, and incubated at 4° C. overnight in the blockingsolution added with the primary antibody. After washing 6 times withPBS, the slide was incubated in the blocking solution containing thesecondary antibody and 1 μg/mL DAPI for 50 min. After washing 6 timeswith PBS, confocal laser scanning microscopic analysis (Olympus FV1000)was performed using Vectashield mounting medium (Vector Laboratories).For 5mC immunofluorescent staining, the slide was treated with 4NHCl/0.1% Triton X at room temperature for 10 min, washed twice with PBSand treated with the blocking solution. The primary antibody andsecondary antibody used are described.

For immunofluorescent staining of cyPGCs, the genital ridge cut out fromE50 (XX) embryo was fixed with 4% PFA/PBS for 15 min at roomtemperature. The tissue was continuously immersed in 10% and 30%sucrose/PBS, embedded in OCT compound (Sakura), freezed and a sectionwith 10 um thickness was produced. The air-dried section was washed 3times with PBS, and incubated in a blocking solution for 1 hr. Thesection was incubated with the primary antibody contained in theblocking solution at room temperature for 2 hr, and washed 4 times withPBS. Then, the section was incubated with the secondary antibodycontained in the blocking solution at room temperature for 50 min,washed 4 times with PBS, and confocal laser scanning microscopicanalysis was performed using Vectashield mounting medium. The antibodiesused are shown in Table 4.

TABLE 4 Antibody Supplier Catalogue# Mouse anti-BLIMP1 R&D SystemsMAB36081 Mouse anti-OCT3/4 SantaCruz sc-5379 Biotechnology Mouseanti-SOX2 R&D Systems MAB2018 Mouse anti-5 Active Motif 39649methylcytosine (5-mC) Rabbit anti-DDX4 Abcam ab13840 Rabbit anti-H3K9Me2Millipore 07-441 Rabbit anti-H3K27Me3 Millipore 07-449 Rabbitanti-TFAP2C Biotechnology sc-8977 SantaCruz Goat anti-SOX17 NeuromicsGT15094 AlexaFluor 488 conjugated Life Technologies A11006 goat anti-ratIgG AlexaFluor 488 conjugated Life Technologies A11001 goat anti-mouseIgG AlexaFluor 568 conjugated Life Technologies A11011 goat anti-rabbitIgG AlexaFluor 633 conjugated Life Technologies A21070 goat anti-rabbitIgG AlexaFluor 633 conjugated Life Technologies A21052 goat anti-mouseIgG

Example 1

Establishment of hiPSCs Having Dual Germ Line Reporter

To examine in vitro induction conditions for differentiation from hiPSCsinto germ cells, hiPSC strain having reporter for BLIMP1 (also known asPRDM1) and TFAP2C (also known as AP2γ) was established. BLIMP1 andTFAP2C were considered to be useful since expression in human germ cellhas been reported (Eckert D, et al., BMC Dev Biol. 8, 106, 2008 andPauls K, et al., Int J Cancer. 115, 470-477, 2005).

In this case, 585A1 and 585B1, which are two line independent straoms ofmale hiPSC derived from peripheral mononuclear blood cells were used ashiPSC (Okita K, et al., Stem Cells. 31, 458-466, 2013). This cell linewas cultured on E8 fragment (rLN511E8) of recombinant laminin511 in agiven medium containing basic fibroblast growth factor (bFGF), wherebysingle cell passage culture with colony forming ability became possible(Nakagawa M, et al., Sci Rep. 4, 3594, 2014). hiPSCs cultured under suchconditions showed more uniform property than culturing under conditionsusing conventional feeder cells, and gene expression property inmultipotency state, which is essentially free of expression of generelating to the primitive pluripotency of mouse (Nakamura T, et al.,Nucleic Acids Res. 43, e60. 2015). Using TALEN (transcriptionactivator-like effector nucleases), dual homologous recombinant iPS cellhaving both BLIMP1-2A-tdTomato and TFAP2C-2A-EGFP allele (alleles) wasisolated. When BLIMP1 and TFAP2C were expressed, the iPS cell showedfluorescence of each of tdTomato and EGFP (FIG. 8, Views A, C, and E).From duble homologous recombinants, BLIMP1-2A-tdTomato; TFAP2C-2A-EGFP585B1-868 strain (hereinafter to be referred to as BTAG 585B1-868),which is a recombinant allele showing both heterozygote and normalkaryotype, was used (FIG. 1, View A, and FIG. 8, View D).

Direct Induction of BTAG Positive Cell from hiPSCs

First, whether BTAG 585B1-868 hiPSCs are directly induced into hPGCLCsunder conditions for inducing mEpiLCs in a transient state extremelysimilar to pre-gastrulation mouse ectoderm into mPGCLCs (Hayashi K, etal., Cell. 146, 519-532, 2011) was examined. BTAG hiPSCs were separatedinto single cells and cultured under floating conditions in GMEM+15%knockout serum replacement (KSR) (GK15) added with major cytokines suchas bone morphogenetic 4 (BMP4), stem cell factor (SCF), leukemiasuppress factor (LIF), and epidermal growth factor (EGF) (3,000cells/cell aggregate) and the like in the presence of a ROCK inhibitor(FIG. 1, View B) (Watanabe K, et al., Nat Biotechnol. 25, 681-686,2007).

In mEpiLCs, cell aggregate of hiPSC did not express BT and AG even after2 days from cytokine stimulation and appeared to be loosely bound byobservation under a fluorescence Anatomy microscope, rather thanstrongly expressing Blimp1 and other PGC specific gene in early stagesof day 2 of stimulation (Hayashi K, et al., Cell. 146, 519-532, 2011)(FIG. 1, View C). However, on day 4, some cells started to express BTAG,and a cell population showed strong BTAG expression on day 6 which wasmaintained at least up to day 8 (FIG. 1, View C). FACS analysissimilarly showed shift of the whole aggregates on day 2 of stimulationto a weak BTAG positive state, about 15% of cells became BTAG positiveon day 4, and BT and AG were upregulated by a similar mechanism. On day6, cell aggregates (about 20%) strongly showing BTAG positive wereobserved which was confirmed to have been maintained at least up to day8 (FIG. 1, View D). However, it should have been noted that aconsiderable number of cells die through induction. Induction wasstarted at 3,000 cells/aggregate, induction efficiency of BTAG positivecells showed the highest value when not less than 10% of KSR was used asthe basal medium for induction (FIG. 8, View F), and the average numberof BTAG positive cells per cell aggregate was about 200 cells/cellaggregate on day 6 of induction (FIG. 1, View E). The above findingsuggests that, different from mEpiSCs and similar to mEpiLCs, hiPSCsshow comparatively strong germ cell formation capacity under appropriateconditions.

The dynamics of the gene expression when BTAG positive cells are inducedfrom hiPSCs were quantitatively examined by PCR. hiPSCs expressmultipotency marker genes POU5F1, NANOG and SOX2 at a high or moderatelevel, whereas show low or no confirmed expression of genes relating toprimitive multipotency such as KLF2, KLF4, TCL1B, TFCP2 L1, ESRRB, andDPPA3 and the like in mouse (FIG. 1, View F), which was consistent withthe report of Nakamura T, et al., Nucleic Acids Res. 43, e60. 2015.However, it should be noted that hiPSCs express ZFP42 and PRDM14 at acomparatively high level (FIG. 1, View F). hiPSCs did not express orshowed only very low expression of genes relating to PGCs (BLIMP1,TFAP2C, NANOS3, DPPA3, DAZL, and DDX4), neuroectoderm (PAX6 and SOX1),mesoderm (T, EOMES, EVX1, SP5, MIXL1, MSX2, and NODAL) and endoderm(GATA4, GATA6, SOX17, and FOXA2) (FIG. 1, View F).

On the other hand, in cell aggregates on day 2 of cytokine stimulation,expression of major multipotency gene increased, expression of primitivepluripotency gene was maintained low, and some genes relating to PGCs(BLIMP1 and TFAP2C), mesoderm (T, EOMES, SP5 and NODAL), and endoderm(GATA4 and SOX17) started to increase (FIG. 1, View F).

Furthermore, BTAG positive cells on day 6 of cytokine stimulationmarkedly increased the expression of POU5F1 and NANOG, but SOX2 did notshow such tendency (FIG. 1, View F). This does not contradict with theresults that hPGCs lack SOX2 expression (de Jong J, et al., J Pathol.215, 21-30, 2008; Perrett R M, et al., Biol Reprod. 78, 852-858, 2008).Mouse early PGC markers such as BLIMP1, TFAP2C and NANOS3 and the likeshowed a high expression level like SOX17, but expression of DPPA3 waslow, and substantial expression of late PGC genes such as DAZL and DDX4and the like could not be confirmed (FIG. 1, View F). In BTAG positivecells, the expression level of T and PRDM14 essential for identificationof mouse PGC was low (FIG. 1, View F). Furthermore, it was found thatBTAG positive cells increase genes relating to primitive pluripotency(KLF4, TCL1B, and TFCP2L1), mesoderm (EVX1 and MSX2) and endoderm(GATA4) of mouse (FIG. 1, View F). While decision is difficult sinceinformation relating to the gene expression property of early hPGCs isnot available, these findings suggest that BTAG positive cells maycorrespond to early hPGCs. These suggest that hPSC may show a state likeformer/proximal-gastrula formation ectoderm having an ability todetermine the germ cell fate.

Example 2 Strong Induction of BTAG Positive Cell Via InitialMesoderm-Like State

While BTAG positive cells are directly induced from hiPSCs, cellaggregates contained a considerable number of dead cells, and the amountof the obtained BTAG positive cells was comparatively small (FIG. 1,View E). Direct induction of germ cell differentiation via formation offloating T cell aggregation may not be an optimal pathway. Thus, theconditions for inducing hiPSCs into an appropriate precursor for germcell differentiation induction were studied. Prior to germ celldifferentiation, based on the finding of the activation of inductioninto mesoderm lineage induced by BMP4 and WNT3 signal in mouse (AramakiS, et al., Dev Cell. 27, 516-529, 2013 and Saitou M, et al., Nature.418, 293-300, 2002), the effect of previously contacting hiPSCs withBMP4, WNT3 and other signaling molecule, which is similar to the use ofvarious extracellular matrix (ECM) constituent components, was examinedfor induction of a precursor for inducing BTAG positive cell.

It was found that, among the conditions studied, a precursor stronglyand stably producing BTAG positive cells is induced by stimulatinghiPSCs in GK15 containing activin A (ACTA, 50 ng/ml) and GSK3 inhibitor(CHIR99021 (3 μM)) on a fibronectin coating plate for about 2 days, andforming floating T cell aggregates under conditions with addition ofBMP4, LIF, SCF, and EGF (FIG. 2, Views A-F, and FIG. 9, Views A-B). Whenstimulated with ACTA and CHIR, hiPSCs are differentiated into cellhaving clear boundary between cells (this cell is referred to as initialmesoderm-like cell (iMeLCs)) (FIG. 2, Views A-B), in the formation offloating cell aggregates accompanying cytokine stimulation, these cellsstart enforced BTAG activation (about 30-40%) on day 2, and the BTAGpositive cells (about 60% on day 4) were maintained at least up to day10 after cytokine stimulation (FIG. 2, Views D-F). The number of BTAGpositive cells per cell aggregate induced from iMeLCs was about 400 to1000 cells/cell aggregate on day 6, which is markedly larger than cellsdirectly induced from hiPSCs (FIG. 2, View F), and cell death was hardlyobserved in the induction of BTAG positive cells from iMeLCs.

Successively, the effect of the related signaling pathway/cultureconditions in the induction of iMeLCs having induction capacity intoBTAG positive cells was evaluated. Like the induction of mEpiLC frommESCs/iPSCs, the efficiency of BTAG positive cell induction largelydepends on the induction time of iMeLCs, and the optimal time for BTAG585B1-868hiPSCs was about 42 hr (FIG. 9, View B). When stimulation withACTA and CHIR was elongated, the property of mesoderm (T, EOMES, EVX1,SP5, MIXL1, and NODAL) and the property of endoderm (GATA4, GATA6,SOX17, and FOXA2) were further increased and BTAG positive cellinduction capacity was lost (FIG. 9, Views B-C). While ACTA (not lessthan 50 ng/ml) and CHIR (3-5 μM) are essential for the induction ofiMeLCs having induction capacity into BTAG positive cells, it was shownthat CHIR is replaceable with WNT3A (not less than 50 ng/ml), and WNTsignaling is essential for iMeLCs induction (FIG. 10, Views A-C). On theother hand, the signaling of BMP4 and bFGF acts negatively to the iMeLCsinduction (FIG. 10, Views D-E). In the induction of iMeLCs, when FGFreceptor (FGFR) signaling is inhibited by a specific inhibitor (PD173074(FGFRi)), the cells in the cell aggregates such as BTAG positive celland the like were more strongly proliferated and survived, and thenumber of BTAG-positive cells per cell aggregate increased (FIG. 10,Views G-H). From these findings, it was clarified that the induction ofiMeLCs having strong BTAG positive cell induction capacity requires aspecific signaling pathway. It was confirmed that iMeLCs is a precursorof mesoderm and embryonic endoderm.

Next, signal pathway necessary for induction of iMeLCs into BTAGpositive cells was examined. While BMP4 is essential for the inductionof BTAG positive cells, SCF, LIF and EGF had a role of maintainingrather than inducing BTAG positive cells in the cell aggregates (FIG.11, Views A-D). Since the effect of BMP4 is blocked by activinreceptor-like kinase2/3 (ALK2/3) inhibitor LDN193189, it was shown thatthe signal from BMP4 goes through ALK2/3 receptor (FIG. 11, View B).From the above, it was confirmed that induction of BTAG positive cellfrom iMeLCs and proliferation/survival thereof requires a signal similarto that of the induction from mEpiLCs to mPGCLCs andproliferation/survival of mPGCLC (Hayashi K, et al., Cell. 146, 519-532,2011).

The dynamics of gene expression in the induction of BTAG positive cellsvia iMeLCs were confirmed by Q-PCR. In iMeLC induction, the expressionlevels of multipotency marker gene (POU5F1, NANOG and SOX2), primitivepluripotency gene (KLF2, KLF4, TCL1B, TFCP2L1, ESRRB and DPPA3), andPGC-related gene (BLIMP1, TFAP2C, NANOS3, DPPA3, DAZL and DDX4) andendoderm-related gene (GATA4 and SOX17) did not change. However, thegenes relating to the development of mesoderm showed a mild increase (T,EOMES, SP5, MIXL1 and NODAL) (FIG. 2, View G, and FIG. 9, View C), andit was shown that stimulation with ACTA and CHIR induces hiPSCs intoinitial mesoderm-like/development initial streak-like state. The geneexpression property of BTAG positive cells induced from iMeLCs wasessentially the same as that of BTAG positive cells directly inducedfrom hiPSCs. That is, the expression of POU5F1 and NANOG was high, SOX2rapidly decreased, initial PGC marker markedly increased like SOX17 andSOX15 (BLIMP1, TFAP2C and NANOS3), and the late PGC genes (DAZL andDDX4) were not substantially expressed (FIG. 2, View G, and FIG. 11,View E).

The expression of OCT4, NANOG, BLIMP1, TFAP2C and SOX17 and thesuppression of SOX2 in BTAG positive cells were also confirmed by FACSand immunofluorescence analysis.

From the above results, it was clarified that hiPSCs are induced intoiMeLCs with increased initial mesoderm genes, and sequentially inducedforcibly into a state potentially similar to BTAG positive cells,initial hPGCs.

Example 3

Transcription of BTAG Positive Cell as hPGCLCs and Analysis of InductionPathway

To further understand the property and induction pathway of BTAGpositive cells, the technique of Nakamura T, et al., Nucleic Acids Res.43, e60. 2015 was used and global transcription profile, and globaltranscription profile relating to the identification of non-humanprimates (Macaca fascicularis) assumed to be similar to human PGCs(global transcription profile of gonad PGCs, and mPGCLCs (mESCs,mEpiLCs, d2, d4, d6 mPGCLCs) were compared in detail for hiPSCs, iMeLCs,BTAG positive cells induced from iMeLCs on day 2 (d2), day 4 (d4), day 6(d6) and day 8 (d8), and BTAG positive cells directly induced fromhiPSCs on day 6 (d6).

To determine the global transcription profile of gonad PGCs of Macacafascicularis (cy), E43, 50, and 51 of Macaca fascicularis embryo (almostcorresponding to mouse E10.5-13.5) were isolated, gonad was removed(FIG. 12, View A), and each gonad was separated into single cells. PGCs(POUF51, NANOG, BLIMP1 and TFAP2C positive)-derived single-cell cDNAswere produced and analysis by RNA-seq was performed (FIG. 12B-D).

By unsupervised hierarchical clustering (UHC), cells relating to theinduction pathway of BTAG positive cells were classified into twoclusters of hiPSCs and iMeLCs each independently constituting asubcluster, and d2-d8 BTAG positive cells having stage dependencysubcluster (FIG. 3, View A). Interestingly, d4 BTAG positive cellsinduced via iMeL state formed subcluster d6 together with BTAG positivecells directly induced from hiPSCs (FIG. 3, View A). This shows that, indirect induction from hiPSCs, BTAG positive cells are formed through apathway similar to BTAG positive cell formation via iMeLCs. By principalcomponent analysis (PCA), iMeLCs were plotted from PC1 negative hiPSCsalong PC2 and PC3 axis, and BTAG positive cells which are almost thesame PC1 positive were plotted in a stage dependent manner and along thePC2 and PC3 axis (FIG. 3, View B). As mentioned above, hiPSCs and iMeLCshave comparatively similar properties, BTAG positive cells haveproperties different from them, and induction of BTAG positive cell is adirectional and progressive process to acquire cell phenotype.

Next, the gene expression properties of cyPGCs and BTAG positive cellswere compared (FIG. 12, View D). cyESCs, CMK9 strain-derived single-cellcDNAs were produced and these cDNAs were subjected to RNA-seq analysis.A gene set showing an increase in cyPGCs as compared to cyESCs wasidentified, and human homologous genes relative to these genes weredetermined. Formation of cluster by d2-d8 BTAG positive cells togetherwith cyPGCs was shown by UHC analysis. Many of the genes co-expressed inBTAG positive cells and cyPGCs related to the gene ontology (GO) termsof “enzyme linked receptor protein signaling pathway”, “stem cellmaintenance”, “reproductive development process” and “gamete production”and the like (FIG. 3, View D). A group of genes not expressed in BTAGpositive cell but expressed in cyPGCs were mainly constituted of thosemainly expressed in late PGCs and having GO terms such as “sexualreproduction”, “sexual differentiation” and the like (FIG. 3, View D).Furthermore, it was clarified by scatter plot analysis that genes up- ordown-regulated in cyPGCs as compared to cyESCs are generally up- ordown-regulated in d6 BTAG positive cells as compared to iMeLCs. However,even though mPGCLC is known to show a transcription profile highlysimilar to mPGCs and have the function as authentic PGCs, this tendencywas very weak when mPGCLCs and mEpiLCs were compared (FIG. 3, View C)(Hayashi K, et al., Science. 338, 971-975, 2012 and Hayashi K, et al.,Cell. 146, 519-532, 2011). Except those relating to late PGC gene, thesefindings demonstrate that BTAG positive cells display gene expressionprofiles similar to those of cyPGCs and that mPGCLCs exhibit total geneexpression significantly different from that of cyPGCs which is assumedto be due to species difference. From the above findings, BTAG positivecells are considered to correspond to initial hPGCs and thereafter showproperty as hPGCLCs.

Next, individual genes that are up- or down-regulated while the cellularstate changes in hPGCLC induction were examined and compared to thegenes in mPGCLC induction (FIG. 4, View A). It was noted that the numberof genes that are up- or down-regulated while the cellular state changesin hPGCLC induction was smaller than the genes regulated during mPGCLCinduction. During iMeLC-d2 hPGCLC change, 991 and 601 genes wererespectively up- or down-regulated, and during mEpiLC-d2 mPGCLC change,1,482 and 870 genes were respectively up- or down-regulated. Duringd2-d4 hPGCLC change, 235 and 126 gene alone were respectively up- ordown-regulated, and during d2-d4 mPGCLC change, 888 and 1,141 genes wererespectively up- or down-regulated (FIG. 4, View A). Many of the genesthat are up-regulated during hiPSC-iMeL change had GO terms of “cellmigration”, “pattern specification process” and the like, and includeCERI, FGF19, NODAL, KITLG, SEMA3C, MIXL1, BMP4, T, EOMES, LEFTY2, FST,PITX2 and the like. On the other hand, Many of the genes that aredown-regulated during hiPSC-iMeL change had GO terms of “chemicalhomeostasis”, “cell adhesion” and the like (FIG. 4, View B).

The genes that are up-regulated during iMeLC-d2 hPGCLC change includethose having a possibility of playing an important role in theidentification of hPGCLC (e.g., TFAP2C, PRDM1, SOX17, SOX15, KLF4, KIT,TCL1A, and DND1) (FIG. 3D and FIG. 4B), and many of them have GO termsof “stem cell maintenance”, “control of cell migration” and the like. Onthe other hand, many of the genes that are down-regulated have GO termsof “pattern specification process”, “neuron development” and the like(FIG. 4, View B). As mentioned above, gene change between d2 and d4hPGCLCs was comparatively small, d4 hPGCLCs down-regulated some genesfor “embryonic morphogenesis” and the total gene expression profile ofd6 and d8 hPGCLCs was essentially the same (FIG. 4, Views A-B).

In hPGCLC and mPGCLC induction, genes expressed at the highest levelamong respective cell types (2-fold or more than other three cell types)were identified (FIG. 4, View C). It has been reported that the majorphenomena of mPGC/PGCLC induction include rapid and powerfulactivation/subsequent suppression of “somatic mesoderm program”(Kurimoto K, et al., Genes Dev. 22, 1617-1635, 2008, Saitou M, et al.,Nature. 418, 293-300, 2002 and Yabuta Y, et al., Biol Reprod. 75,705-716, 2006). The gene (n=756) of the maximum group was found to bemost frequently expressed in d2 mPGCLCs (d2 mPGCLC gene). These genesshowed remarkably high P values, had many GO terms such as “embryonicmorphogenesis” and “pattern identification process”, and the like andthe majority thereof were suppressed by d4 rnPGCLCs (FIG. 4, Views C-D).The number of genes most frequently expressed in d2 hPGCLCs (d2 hPGCLCgene) was comparatively small (104), and the P value was not high.However, many genes had GO terms mainly including “cellular componentmorphogenesis” and “neuron differentiation” and were graduallysuppressed by d4 and d6 hPGCLCs (FIG. 4, Views C-D).

Many of the human homologous genes of d2 mPGCLC gene showed relativelyconstant expression in hPGCLC induction (no expression or expression atthe same level). On the other hand, only a small number of mousehomologous genes of d2 hPGCLC gene showed transient upregulation in d2mPGCLC gene (FIG. 4, Views E and G). Therefore, only 16 genes containingWNT5A, WNT5B, CFC1, EVX1, SNAI2 and CDX2 but free of Hox gene werecommon in d2 hPGCLC gene and d2 mPGCLC gene (FIG. 4, View E, and FIG.12, View F).

From the above results, transcription programming in hPGCLC and mPGCLCinduction was shown and it was shown that hPGCLC induction did not showmarked activation/subsequent suppression of “somatic mesoderm program”.

Successively, to investigate the precursor state of hPGCLC induction,the relationship between hiPSCs, iMeLCs, mESCs, mEpiLCs, and mEpiSCs wasexamined. Since the overall transcriptional state varies betweenspecies, direct comparison between human and mouse cells is notpossible. Thus, representative genes of mice having major functionrelating to inner cell mass (ICM)/primitive pluripotency,pre-gastrulation ectoderm, mesoderm, and endoderm formation wereselected and expression profiles in these cells were examined. As shownin FIG. 4, View H, genes relating to ICM/primitive pluripotency werehighly expressed in mESCs, decreased in mEpiLCs, and further decreasedin mEpiSCs, but genes relating to ectoderm increased in both mEpiLCs andmEpiSCs. Genes relating to mesoderm and endoderm were generally low inmESCs and mEpiLCs, but increased to some extent in mEpiSCs (FIG. 4, ViewH). These data are not consistent with the idea that mESCs, mEpiLCs andmEpiSCs have properties resembling ICM/initial ectoderm,pre-gastrulation ectoderm, and post-gastrulation ectodermprecastembryonic ectoderm, and posterior embryonic ectoderm respectively.

The expression patterns of genes relating to ICM/primitive pluripotencyin hiPSCs and iMeLCs were similar to mEpiLCs, but not similar to mESCs(FIG. 4, View H). About half of the genes selected in relation to mousepre-gastrulation ectoderm were strongly expressed in hiPSCs and iMeLCsbut the remaining half did not show remarkable expression, which isconsidered to be caused by species differences. Similar to the Q-PCRanalysis (FIG. 1, View F; FIG. 2, View G; and FIG. 9, View C), somegenes of mesoderm rather than endoderm formation increased in iMeLCs butnot in hiPSCs (FIG. 4, View H).

From the above results, hiPSCs cultured under this condition hadproperties most similar to mEpiLCs out of mESCs, mEpiLCs and mEpiSCs,regardless of the species differences, and it was suggested that theycan be reproductive cells by human-specific pathway.

Next, the epigenetic profile of hPGCLCs was examined. Like mousemigrating PGC, in mPGCLCs, histone H3 lysine 9 dimethylation (H3K9me2)level decreased, histone H3 lysine 27 trimethylation (H3K27me3) levelincreased, and DNA methylation [5-methylcytosine (5mC)] level decreased,as compared to mEpiLCs, but parental imprints were maintained (HayashiK, et al., Cell. 146, 519-532, 2011 and Kurimoto K, et al., Genes Dev.22, 1617-1635, 2008). Next, H3K9me2, H3K27me3 and 5mC levels werecompared between hiPSCs and d8 hPGCLCs by immunofluorescence analysis.While H3K9me2 level in d8 hPGCLCs was lower than that in hiPSCs,H3K27me3 level varied. While hPGCLCs showing higher H3K27me3 levels werepresent, those showing H3K27me3 level similar to hiPSCs were alsopresent (FIG. 13, View A). Furthermore, 5mC level in d8 hPGCLCs waslower than hiPSCs (FIG. 13, View A).

To elucidate the mechanism of epigenetic reprogramming by hPGCLCs, theexpression of epigenetic modification factor relating to hPGCLCinduction was examined by reference to gonad cyPGCs. Among the moleculesincluded in DNA methylation, DNMT3B rapidly decreased in hPGCLCs, butthe expression of DNMT1 and UHRF1 was maintained. DNMT3A, DNMT3B andUHRF1 decreased in cyPGCs (FIG. 13, View C). Among the moleculesincluded in DNA methylation, TET1 was expressed in a relatively constantamount, but other genes were not expressed or low in hPGCLC inductionand cyPGCs (FIG. 13, View C). EHMT2, which is the major enzyme ofH3K9me2, was suppressed by hPGCLCs and cyPGCs, and several H3K9me2demethylases (KDM1A, KDM3A, KDM3B) were expressed (FIG. 13, View C).Among the molecules included in H3K27me3, EZH2 and SUZ12 showed aconstant expression level during PGCLC induction, but EED was suppressedin hPGCLCs. On the other hand, in cyPGCs, EZH2, EED, and SUZ12 werestrongly expressed (FIG. 13, View C).

Example 4

Search for Surface Marker of hPGCLCs

To induce and identify hPGCLCs from hiPSCs without fluorescencereporters, surface markers that identify hPGCLCs were searched for.Among the screened several surface markers, a combination of [PECAM(CD31), INTEGRINα6 (CD49f), INTEGRINβ3 (CD61), KIT (CD117), EpCAM,PODOPLANIN, and TRA1-81], EpCAM (APC-A channel) and INTEGRINα6 (HorizonV450-A channel) separated day 6 cell aggregates induced from BTAG585B1-868 hiPSCs into three different populations. That is, they wereseparated into high EpCAM and high INTEGRINα6 (P3 gate), high EpCAM andlow INTEGRINα6 (P4 gate), low EpCAM and low INTEGRINα6 (P5 gate) (FIG.5, View A). The high EpCAM and high INTEGRINα6 population (P3 gate) wasalmost the same in BTAG positive hPGCLCs (about 98.9% of high EpCAM andhigh INTEGRINα6 cells were BTAG positive cells; BT: PE-Texas Red-Achannel; AG: FITC-A channel). Other populations were substantiallynegative/weakly positive for BTAG (FIG. 5, View A). On day 2 ofinduction, the high EpCAM and high INTEGRINα6 population wasidentifiable (about 37%), and substantially the same as the BTAGpositive population (about 98% of high EpCAM and high INTEGRINα6 cellswere BTAG positive). Thereafter, they remained at least up to day 8(FIG. 5, View B).

To investigate whether hPGCLCs can be isolated using surface markers,independent reporter-free strain 585A1 hiPSCs were induced into hPGCLCsvia iMeLCs. As shown in FIG. 5, View C, on day 2 of induction, a highEpCAM and high INTEGRINα6 population (about 21%) emerged in cellaggregates, and this population increased up to day 4 (about 31%) andday 6 (about 32%). To determine whether this population is hPGCLCs,total RNA was extracted from the population on day 6 of induction andthe global transcription profile was analyzed by RNA-seq. As shown inFIG. 5, View D, UHC analysis indicate that high EpCAM and highINTEGRINα6 cells on day 6 confluence with BTAG positive hPGCLCs on days6 and 8 and it was confirmed that high EpCAM and high INTEGRINα6 cellson day 6 were hPGCLCs.

Example 5

Important Function of BLIMP1 in Identifying hPGCLC

The role of identifying hPGCLC of BLIMP1 which is an importanttranscription factor for identifying mouse PGC was investigated. Usingthe TALEN homologous recombination strategy, a hiPSC strain havingTFAP2C-2A-EGFP allele was produced (AG 585B1-17, 19), and exon 4 ofBLIMP1 gene was replaced with tdTomato so that tdTomato would beexpressed and BLIMP1 would be knocked out with the target allele (FIG.6, View A, and FIG. 14, Views A-B). Similar to one replaced by otherallele with a frameshift deletion and tdTomato and containing one BLIMPallele (BLIMP1 homozygously knocked out clones: BLIMP1^(−/−)), severalclones knocked out by heterozygous targeting of BLIMP1 were isolated(BTAG; BLIMP1^(+/−)) (FIG. 6, View A, and FIG. 14, View B).

AG; BLIMP1^(+/+), BTAG; BLIMP1^(+/−), and BTAG; BLIMP1^(−/−) hiPSCs wereinduced into iMeLCs, and further induced into hPGCLCs. It was confirmedthat AG positive cells induced from BTAG; BLIMP1^(−/−) hiPSCs do notexpress BLIMP1 (FIG. 6, View B). As shown in FIG. 6, Views C-E, AG;BLIMP1^(+/+) hiPSCs were strongly induced into AG positive hPGCLCs andmaintained as hPGCLCs (d2: about 69.3%; d4: about 52.8%; d6: about32.2%; d8: about 32.2%). In contrast, BTAG; BLIMP1^(−/−) hiPSCs werecomparatively efficiently induced into BTAG positive cell on day 2 ofinduction (about 47.4%), but the cell number of BTAG positive rapidlydecreased on day 4 (about 18.8%), and these cells almost disappeared onday 6 (about 1.6%) (FIG. 6, View C-E). From this, it was shown thatBLIMP1 is essential for identification/maintenance of hPGCLCs. Similarto the results of capacity-dependent function of mouse Blimp1 (OhinataY, et ai., Nature. 436, 207-213, 2005; Vincent S D, et al., Development.132, 1315-1325, 2005), BTAG; BLIMP1^(+/−) hiPSCs showed an intermediatephenotype between wild-type and BTAG; BLIMP1^(−/−) hiPSCs. That is, theinduction rate of BTAG positive cells on days 2, 4, 6, and 8 wasrespectively about 49.6%, about 27.0%, about 8.1% and about 4.6% (FIG.6, Views C-E). The same results were obtained when other independentstrain BTAG; BLIMP1^(−/−) hiPSCs were used.

To investigate the role of BLIMP1 in hPGCLC induction, RNA was extractedfrom d2 and d4 (BT) AG positive cells induced from AG; BLIMP1^(+/+) andBTAG; BLIMP1^(−/−) hiPSCs and the expression of major genes was analyzedby Q-PCR (FIG. 6, View F). BLIMP1^(−/−); BTAG positive cells increasedTFAP2C, but an increase of gene NANOS3, KLF4, TFCP2L1, and TCL1B and thelike was suppressed (FIG. 6, View F). BLIMP1^(+/−); BTAG positive cellsdid not maintain T and MIXL1 but did not suppress EVX1 and SP5, and aneffect on EOMES and MSX2 was not found (FIG. 6, View F). This indicatesthat BLIMP1 exerts other effect on genes relating to mesodermdevelopment. Similarly, in BLIMP1^(−/−); BTAG positive cells, GATA4suppressed suppression but a clear effect on SOX17, GATA6, and FOXA2 wasnot found (FIG. 6, View F). Particularly, BLIMP1^(−/−); BTAG positivecells did not suppress DNMT3B (FIG. 6, View F).

The transcriptome of BLIMP1^(−/−); BTAG positive cells was compared withwild-type and BLIMP1^(+/−) hPGCLCs by RNA-seq. While BLIMP1^(−/−); BTAGpositive cells acquired properties similar to those of d2 hPGCLCs, UHCand PCA analysis showed that further differentiation towards d4 PGCLCstate did not proceed (FIG. 7, Views A-B). To examine faulty inBLIMP1^(−/−) cells, genes up- or down-regulated in d2 and d4BLIMP1^(−/−); BTAG positive cells were respectively compared with d2 andd4 PGCLCs. Many of the genes (104 genes) increased in d2 BLIMP1^(−/−);BTAG positive cells had GO terms of “neuron differentiation”, “gastrulaformation” and “embryonic morphogenesis”, and many of the genes (692genes) increased in d4 BLIMP1^(−/−); BTAG positive cells had higher Pvalues and similar GO terms. This indicates that BLIMP1 functions tosuppress such development programs in hPGCLCs (FIG. 7, Views C-D). Incontrast, the number of genes suppressed in BLIMP1^(−/−); BTAG positivecells was smaller (61 and 300 genes for d2 and d4, respectively), andmany GO terms related to apoptosis and cell cycle. This suggests amisregulation of basic cellular properties in BLIMP1^(−/−); BTAGpositive cells (FIG. 7, Views C-D).

the genes up- or down-regulated in BLIMP1^(−/−); BTAG positive cellsshowed different expression patterns in hPGCLC induction andparticularly, in BLIMP1^(+/−); BTAG positive cells, intermediate geneexpression pattern between wild-type and BLIMP1^(−/−) cells was shown(FIG. 7, View E). The genes relating to “neuron differentiation” and“embryonic morphogenesis” and increased in d2 BLIMP1^(−/−); BTAGpositive cells expressed little or did not express in wild-type d2PGCLCs (FIG. 7, View E). From these data, it was clearly shown thatBLIMP1 controls the expression level of genes widely in a dose-dependentmanner in hPGCLC induction.

Very many genes relating to “neuron differentiation” and the related GOterms were found in genes that increase in d2 and d4 BLIMP1^(−/−); BTAGpositive cells (FIG. 7, View D). Thus, the dynamics of gene expressionof “neuronal differentiation” in wild-type hPGCLC induction and theeffect of BLIMP1 deficiency in this expression were investigated. Asshown in FIG. 7, View F, the genes of “neuron differentiation” areclassified into several expression categories. The genes that increasein BLIMP1^(−/−) cells were abundant in the expression categoriesrelating to upregulation in d2 hPGCLCs (18/39, about 46%) and expressioncategories relating to downregulation (6/23, about 26%). 15 genes for“neuronal differentiation” whose expression does not change remarkablyin wild-type hPGCLC induction increased in d2 or d4 BLIMP1^(−/−) cells(FIG. 14, View E).

From the above results, it was suggested that the main function ofBLIMP1 is to suppress the “neuronal differentiation” gene that increasedin d2 hPGCLCs to a moderate level, and to appropriately suppress suchgenes that decrease in d2 hPGCLCs.

INDUSTRIAL APPLICABILITY

According to the differentiation induction method of the presentinvention, human pluripotent stem cells can be induced to differentiateinto human PGC-like cells with high efficiency and good reproducibility.In addition, using the cell surface marker of the present invention,human PGC-like cells can be isolated and purified efficiently.Therefore, germ cell differentiation determination pathway from humanpluripotent stem cells can be functionally reconstituted in vitro, andthe present invention is markedly useful for elucidating thedevelopmental mechanism of germ cells in human and for establishingdiagnosis/treatment of diseases caused by defects in human germ cells.

The contents disclosed in any publication stated in the presentspecification, including patents, patent applications and scientificliteratures, are hereby incorporated in their entireties by reference,to the extent that they have been disclosed herein.

This application is based on a patent application No. 2015-130501 filedin Japan (filing date: Jun. 29, 2015), the contents of which areincorporated in full herein.

1. A method for sorting a human primordial germ cell-like (PGC-like)cell comprising selecting a cell positive to at least one cell surfacemarker selected from the group consisting of PECAM (CD31), INTEGRINα6(CD49f), INTEGRINβ3 (CD61), KIT (CD117), EpCAM, PODOPLANIN and TRA1-81.2. The method according to claim 1 wherein said selection is performedby selecting a double positive cell of INTEGRINα6 (CD49f) and EpCAM.